Rapid-acting insulin compositions

ABSTRACT

The invention is a composition of human insulin or insulin analog that includes specific concentrations of citrate, chloride, in some cases including the addition of sodium chloride, zinc and, optionally, magnesium chloride and/or surfactant, and that has faster pharmacokinetic and/or pharmacodynamic action than commercial formulations of existing insulin analog products.

The present invention is a pharmaceutical insulin composition for use inthe treatment of diabetes to counteract post-prandial blood glucoseexcursions and for acute treatment of hyperglycemia. The compositionincludes human insulin or an insulin analog, and specific concentrationsof citrate, chloride, in some cases including the addition of sodiumchloride, zinc and, optionally, magnesium chloride and/or surfactant.The composition has a faster uptake of insulin from injection sites thanexisting commercial insulin compositions. The composition is useful forproviding meal-time insulin activity or an acute treatment forhyperglycemia when insulin is needed.

The time-action profile of insulin is important for controllingpost-prandial blood glucose levels. In healthy individuals, the pancreassecretes a spike of insulin in response to absorbed food, which resultsin increased blood insulin levels within several minutes. In individualswith type 1 diabetes and in certain individuals with type 2 diabetes,insulin must be administered. However, administered insulin enters bloodslowly. Inadequate release of insulin and onset which is too slow at thebeginning of a meal leads to hyperglycemia during or immediately afterthe meal. Too long duration of action and excessive insulin betweenmeals causes postprandial hypoglycemia.

There have been previous efforts to reduce the time-action of insulinproducts. Early efforts to develop such products included thedevelopment of novel “rapid-acting” insulin analogs, like insulin lispro(HUMALOG®), insulin aspart (NOVOLOG®), and insulin glulisine (APIDRA®).Insulin lispro and insulin aspart achieve rapid action through changesin the amino acid sequences from the sequences of the amino acid chainsin human insulin. Insulin glulisine also includes changes in thesequences of the amino acid chains in human insulin, and also lacks zincand does not form stabilizing hexamers. The rapid-acting insulin analogsbecame available in the 1990s and early 2000s. Even with so-calledrapid-acting insulin analogs, however, the maximum insulin level is notreached until 50-90 minutes following the injection. This is slower thaninsulin is released by a normally functioning pancreas and does notalways match carbohydrate absorption profiles.

Another avenue to achieve rapid action that has been explored is the useof ingredients or excipients which improve the time action profile ofinsulin when provided in combination with insulin.

For example, US2013/0231281 discloses aqueous compositions comprising aninsulin and particular oligosaccharides, either alone or in combinationwith a polyanionic compound. The polyanionic compound is an anionicpolymer selected from the group consisting of selected from the groupconsisting of dextranmethylcarboxylic acid, polyglutamic acid,polyaspartic acid, PAA (polyacrylic acid), alginate, hyaluronic acid,polymers based on glucuronic acid or based on galacturonic acid andtheir salts, or is an anionic compound selected from the groupconsisting of citric acid, aspartic acid, glutamic acid, malic acid,tartaric acid, succinic acid, adipic acid, oxalic acid, triphosphate,polyphosphate, and their salts. In one embodiment, the polyanioniccompound is sodium citrate. The polyanionic compounds are said toimprove the performance of the oligosaccharide-containing compositionseven when the oligosaccharide alone or the polyanionic compound alonehas no effect on time action.

No insulin time action results for compositions containing citrate butlacking an oligosaccharide are disclosed.

In addition, US2012/0178675 and US2014/0113856 disclose compositionscontaining insulin in combination with a zinc chelator such asethylenediaminetetraacetic acid (EDTA) and what are described as“dissolution/stabilization” agents, such as citric acid or sodiumcitrate. US2014/0113856 states that early clinical trials on suchcompositions showed injection site discomfort, and purports to increaseinjection site tolerability through the addition of a magnesiumcontaining compound. Such magnesium compounds include inorganic salts,such as magnesium hydroxide, magnesium sulfate, magnesium chloride,magnesium bromide, magnesium iodide, magnesium pyrophosphate, magnesiumsulfate heptahydrate and magnesium oxide; and organic salts, such asmagnesium EDTA, magnesium lactate, magnesium aspartate, magnesiumacetate, magnesium carbonate, magnesium citrate, and magnesiumgluconate. US2012/0178675 and US2014/0113856 did not disclose insulintime action results for compositions containing citric acid or citratebut not containing EDTA. A 2012 poster by the assignee of thoseapplications, however, did compare the time action profile of an insulinlispro composition containing citrate and no EDTA to compositionscontaining varying concentrations of EDTA and citrate, and that posterstates that “[c]itrate alone in the formulation is insufficient toimprove the [insulin lispro] absorption profile;” and that “[b]othchelation of zinc with EDTA (to hasten disassembly of the less stableinsulin analog hexamer) and citrate (to mask the surface charge andprevent re-aggregation) are required above a threshold concentration toenhance the subcutaneous rate of absorption.” Roderike Pohl, et al.,“Development of Ultra-Rapid-Acting Prandial Insulin Analogs RequiresChelation of Zinc Ions and Charge Masking to Increase the Rate ofSubcutaneous Absorption,” (EASD 2012).

US2015/0065423 describes compositions comprising a peptide and avasodilatory agent (e.g., nitroglycerin) and, optionally, what aredescribed as “charge masking agents,” such as citrate, adiketopiperazine, a diketopiperazine derivative, EDTA, di-argininepiperazine, a diarginine piperazine salt, a di-arginine piperazineisomer, a di-arginine piperazine ester and any combination thereof. Nocompositions containing citrate but lacking a vasodilatory agent aredisclosed.

The prior art collectively does not teach an ultra-rapid insulinformulation with the excipients and specific concentrations thereofincluded in the present invention.

Despite the efforts described above, there remains a need for insulincompositions, intended for use at meal-time, that have more rapid uptakeof insulin into the blood from the injection site; more rapid onset ofaction than existing insulin products; and chemical and physicalstability during storage. The present invention seeks to providecompositions which meet these needs.

The present inventors have surprisingly found that compositionscontaining certain concentrations of citrate have more rapid uptake ofinsulin into the blood and/or onset of action than existing commerciallyavailable insulin compositions. The chemical and physical stability ofthe compositions under certain conditions is maintained, withouteliminating the improvements in time action, by including in thecompositions certain concentrations of stabilizing agents, such as zinc,sodium chloride, and, optionally, magnesium chloride and/or surfactant.

Accordingly, the present invention provides pharmaceutical compositionscomprising insulin in a concentration of about 100 to about 200 IU/mL,citrate in a concentration of about 15 to about 35 mM, zinc in aconcentration of about 0.2 to about 0.8 mM, and a preservative. Incertain embodiments, the pharmaceutical compositions further comprisemagnesium chloride in a concentration of up to about 5 mM.

In another embodiment, the present provides generally compositionscomprising an insulin and specific concentrations of citrate andstabilizing agents.

In certain embodiments, the present invention provides pharmaceuticalcompositions comprising an insulin; citrate, in a concentration fromabout 10 to about 30 mM; zinc, in a concentration from about 0.2 toabout 2 mM; magnesium, in a concentration from about 1 to about 15 mM;total chloride in a concentration from about 10 to about 60 mM; asurfactant, in a concentration from about 0.001 to about 0.2% w/v; and apreservative.

In certain embodiments, the present invention provides pharmaceuticalcompositions comprising an insulin; citrate, in a concentration fromabout 10 to about 30 mM; zinc, in a concentration from about 0.2 toabout 2 mM; magnesium, in a concentration from about 1 to about 15 mM;sodium chloride in a concentration from about 1 to about 50 mM; asurfactant, in a concentration from about 0.001 to about 0.2% w/v; and apreservative.

In certain embodiments, pharmaceutical compositions of the presentinvention comprise insulin lispro, in a concentration from about 100 toabout 500 IU/mL; citrate, in a concentration from about 10 to about 25mM; magnesium, in a concentration of about 2 to about 9 mM; zinc, in aconcentration from about 0.3 to about 1.3 mM; total chloride, in aconcentration from about 15 to about 35 mM; poloxamer 188, in aconcentration from about 0.03 to about 0.12% w/v; and metacresol, in aconcentration of about 2.8 to about 3.5 mg/mL; and wherein the pH of thecomposition is from about 7.0 to 7.8.

In certain embodiments, pharmaceutical compositions of the presentinvention comprise insulin lispro, in a concentration from about 100 toabout 500 IU/mL; citrate, in a concentration from about 10 to about 25mM; magnesium, in a concentration of about 2 to about 9 mM; zinc, in aconcentration from about 0.3 to about 1.3 mM; sodium chloride, in aconcentration from about 1 to about 25 mM; poloxamer 188, in aconcentration from about 0.03 to about 0.12% w/v; and metacresol, in aconcentration of about 2.8 to about 3.5 mg/mL; and wherein the pH of thecomposition is from about 7.0 to 7.8.

In certain embodiments, pharmaceutical compositions of the presentinvention comprise insulin lispro, in a concentration from about 100 toabout 200 IU/mL; citrate, in a concentration from about 15 to about 25mM; magnesium chloride, in a concentration of about 3 to about 8 mM;zinc, in a concentration from about 0.3 to about 1.1 mM; total chloride,in a concentration from about 20 to about 25 mM; poloxamer 188, in aconcentration from about 0.06 to about 0.09% w/v; and metacresol, in aconcentration of about 2.8 to about 3.5 mg/mL; and wherein the pH of thecomposition is from about 7.0 to 7.8.

In certain embodiments, pharmaceutical compositions of the presentinvention comprise insulin lispro, in a concentration from about 100 toabout 200 IU/mL; citrate, in a concentration from about 15 to about 25mM; magnesium chloride, in a concentration of about 3 to about 8 mM;zinc, in a concentration from about 0.3 to about 1.1 mM; sodiumchloride, in a concentration from about 1 to about 20 mM; poloxamer 188,in a concentration from about 0.06 to about 0.09% w/v; and metacresol,in a concentration of about 2.8 to about 3.5 mg/mL; and wherein the pHof the composition is from about 7.0 to 7.8.

In certain embodiments, pharmaceutical compositions of the presentinvention comprise insulin lispro, in a concentration from about 100 toabout 200 IU/mL; citrate, in a concentration from about 15 to about 25mM; magnesium chloride, in a concentration of about 3 to about 8 mM;zinc, in a concentration from about 0.6 to about 1.1 mM; total chloride,in a concentration from about 20 to about 25 mM; poloxamer 188, in aconcentration from about 0.06 to about 0.09% w/v; and metacresol, in aconcentration of about 2.8 to about 3.5 mg/mL; and wherein the pH of thecomposition is from about 7.0 to 7.8.

In certain embodiments, pharmaceutical compositions of the presentinvention comprise insulin lispro, in a concentration from about 100 toabout 200 IU/mL; citrate, in a concentration from about 15 to about 25mM; magnesium chloride, in a concentration of about 3 to about 8 mM;zinc, in a concentration from about 0.6 to about 1.1 mM; sodiumchloride, in a concentration from about 1 to about 20 mM; poloxamer 188,in a concentration from about 0.06 to about 0.09% w/v; and metacresol,in a concentration of about 2.8 to about 3.5 mg/mL; and wherein the pHof the composition is from about 7.0 to 7.8.

In certain embodiments, pharmaceutical compositions of the presentinvention comprise insulin lispro, in a concentration of 100 IU/mL;citrate, in a concentration of about 25 mM; zinc, in a concentration ofabout 0.6 to about 0.9 mM; magnesium chloride, in a concentration ofabout 5 mM; total chloride, in a concentration of about 20 to about 25mM; poloxamer 188, in a concentration of about 0.09% w/v; andmetacresol, in a concentration of about 3.15 mg/mL; and wherein the pHof the composition is about 7.4.

In certain embodiments, pharmaceutical compositions of the presentinvention comprise insulin lispro, in a concentration of 100 IU/mL;citrate, in a concentration of about 25 mM; zinc, in a concentration ofabout 0.6 to about 0.9 mM; magnesium chloride, in a concentration ofabout 5 mM; sodium chloride, in a concentration of about 1 to about 20mM; poloxamer 188, in a concentration of about 0.09% w/v; andmetacresol, in a concentration of about 3.15 mg/mL; and wherein the pHof the composition is about 7.4.

In certain embodiments, pharmaceutical compositions of the presentinvention comprise insulin lispro, in a concentration of 100 IU/mL;citrate, in a concentration of about 25 mM; zinc, in a concentration ofabout 0.9 mM; magnesium chloride, in a concentration of about 5 mM;total chloride, in a concentration of about 20-25 mM; poloxamer 188, ina concentration of about 0.09% w/v; and metacresol, in a concentrationof about 3.15 mg/mL; and wherein the pH of the composition is about 7.4.

In certain embodiments, pharmaceutical compositions of the presentinvention comprise insulin lispro, in a concentration of 100 IU/mL;citrate, in a concentration of about 25 mM; zinc, in a concentration ofabout 0.9 mM; magnesium chloride, in a concentration of about 5 mM;sodium chloride, in a concentration of about 1 to about 20 mM; poloxamer188, in a concentration of about 0.09% w/v; and metacresol, in aconcentration of about 3.15 mg/mL; and wherein the pH of the compositionis about 7.4.

In certain embodiments, pharmaceutical compositions of the presentinvention comprise insulin lispro, in a concentration of about 200IU/mL; citrate, in a concentration from about 15 to about 25 mM;magnesium chloride, in a concentration of about 3 to about 8 mM; zinc,in a concentration from about 0.8 to about 1.1 mM; total chloride, in aconcentration from about 20 to about 25 mM; poloxamer 188, in aconcentration from about 0.06 to about 0.09% w/v; and metacresol, in aconcentration of about 2.8 to about 3.5 mg/mL; and wherein the pH of thecomposition is from about 7.0 to 7.8.

In certain embodiments, pharmaceutical compositions of the presentinvention comprise insulin lispro, in a concentration of about 200IU/mL; citrate, in a concentration from about 15 to about 25 mM;magnesium chloride, in a concentration of about 3 to about 8 mM; zinc,in a concentration from about 0.8 to about 1.1 mM; sodium chloride, in aconcentration from about 1 to about 20 mM; poloxamer 188, in aconcentration from about 0.06 to about 0.09% w/v; and metacresol, in aconcentration of about 2.8 to about 3.5 mg/mL; and wherein the pH of thecomposition is from about 7.0 to 7.8.

In addition, the present invention also provides a method of treatingdiabetes comprising administering to a human in need thereof aneffective dose of a pharmaceutical composition of the present invention.

In addition, the present invention provides a pharmaceutical compositionof the present invention for use in therapy. More particularly, thepresent invention provides a pharmaceutical composition of the presentinvention for use in the treatment of diabetes. The present inventionalso provides the use of a pharmaceutical composition of the presentinvention in the manufacture of a medicament for the treatment ofdiabetes.

In addition, the present invention provides an article of manufacturecomprising a pharmaceutical composition of the present invention. Inparticular, the present invention provides an article of manufacturecomprising a pharmaceutical composition of the present invention whereinthe article of manufacture is a multi-use vial. In particular, thepresent invention provides an article of manufacture comprising apharmaceutical composition of the present invention wherein the articleof manufacture is a re-usable pen injector. In particular, the presentinvention provides an article of manufacture comprising a pharmaceuticalcomposition of the present invention wherein the article of manufactureis a pump device for continuous subcutaneous insulin infusion therapy.

In an embodiment, the insulin is selected from the group consisting ofhuman insulin, or a rapid-acting structural variant, mutant, or analogof human insulin, such as insulin lispro, insulin aspart or insulinglulisine. In a preferred embodiment, the insulin is insulin lispro.

In an embodiment, the preservative is selected from the group consistingof phenol and meta-cresol. Preferably, the preservative is meta-cresol.In an embodiment, the meta-cresol concentration is from about 2.5 mg/mLto about 3.8 mg/mL. Preferably, the meta-cresol concentration is about3.15 mg/mL.

In an embodiment, the composition further comprises a tonicity agent. Inan embodiment, the tonicity agent is glycerol. In certain embodiments,the glycerol concentration is from about 1 to about 16 mg/mL.

In an embodiment, the composition further comprises a buffer. In certainembodiments, the buffer is sodium phosphate.

In certain embodiments the composition further comprises an additionalstabilizing agent. In certain embodiments the composition furthercomprises a surfactant.

In certain embodiments, the composition does not include any additionalchelating agent, such as EDTA, any additional vasodilatory agent, suchas nitroglycerin, and/or any oligosaccharides.

In an embodiment, the pH of the composition is from about 7.0 to about7.8. In certain preferred embodiments, the pH of the composition is fromabout 7.3 to about 7.5. In certain preferred embodiments, the pH of thecomposition is about 7.4.

In certain embodiments, the pharmaceutical composition provides for anuptake of insulin into the blood and/or onset of action that is at least10%, at least 20%, at least 30%, at least 40%, or at least 50% morerapid than for compositions of rapid-acting insulin analogs which do notcontain citrate, when measured by one or more pharmacokinetic orpharmacodynamic parameters relevant to time action, such as: time tomaximum insulin concentration (Tmax); time to reach one half of themaximum insulin concentration (early ½ Tmax); time to reach one half ofthe maximum insulin concentration during the declining phase of theconcentration-over-time curve (late ½ Tmax); time between early and late½ Tmax (Tmax spread); percentage of total insulin dose absorbed atdifferent times based on fractional area under the insulin concentrationcurve (e.g., AUC_(0-30min), AUC_(0-60min), AUC_(0-120min),AUC_(0-180min)), time to reach one half of the total insulinconcentration (T50); time to reach maximal glucose infusion rate(GIRmax), time to reach one half of the maximum glucose infusion rate(early ½ GIRmax); time to reach one half of the maximum glucose infusionrate during the declining phase of the concentration-over-time curve(late ½ GIRmax); percentage of total glucose infused at different timesbased on fractional area under the GIR curve (e.g., GIR_(0-30min),GIR_(0-60min), GIR_(0-120min), GIR_(0-180min)).

In certain embodiments, the pharmaceutical composition provides for anuptake of insulin into the blood and/or onset of action that is between10% and 50% more rapid than for compositions of rapid-acting insulinanalogs which do not contain citrate, when measured by one or morepharmacokinetic or pharmacodynamic parameters relevant to time action,such as those described above.

In certain embodiments, the pharmaceutical composition is stable toallow for storage of at least 24 months at 2-8° C. In certainembodiments, the pharmaceutical composition is stable to allow for up to28 days in-use at temperatures of up to 30° C. for vials or cartridgesin re-usable pen injectors. In certain embodiments, the pharmaceuticalcomposition is stable to allow for storage of at least 36 months at 2-8°C. In certain embodiments, the pharmaceutical composition is stable toallow for up to 32 days in-use at temperatures of up to 30° C. for vialsor cartridges in re-usable pen injectors. In certain embodiments, thecomposition is stable to allow for use in a pump device for continuoussubcutaneous insulin infusion therapy for up to 7 days.

In an embodiment, the pharmaceutical composition comprises insulinlispro, in a concentration of about 100 IU/mL; citrate, in aconcentration of about 15 mM; zinc, in a concentration of about 0.3 mM;sodium chloride, in a concentration of about 15 mM; magnesium chloride,in a concentration of about 5 mM; metacresol, in a concentration ofabout 3.15 mg/mL; and glycerol, in a concentration of about 7.6 mg/mL.

In another embodiment, the pharmaceutical composition comprises insulinlispro, in a concentration of about 100 IU/mL; citrate, in aconcentration of about 25 mM; zinc, in a concentration of about 0.3 mM;sodium chloride, in a concentration of about 15 mM; magnesium chloride,in a concentration of about 5 mM; metacresol, in a concentration ofabout 3.15 mg/mL; and glycerol, in a concentration of about 4.5 mg/mL.

In another embodiment, the pharmaceutical composition comprises insulinlispro, in a concentration of about 100 IU/mL; citrate, in aconcentration of about 35 mM; zinc, in a concentration of about 0.3 mM;sodium chloride, in a concentration of about 23 mM; magnesium chloride,in a concentration of about 5 mM; and metacresol, in a concentration ofabout 3.15 mg/mL.

In another embodiment, the pharmaceutical composition comprises insulinlispro, in a concentration of about 100 IU/mL; citrate, in aconcentration of about 25 mM; zinc, in a concentration of about 0.3 mM;sodium chloride, in a concentration of about 25 mM; and metacresol, in aconcentration of about 3.15 mg/mL.

In another embodiment, the pharmaceutical composition comprises insulinlispro, in a concentration of about 100 IU/mL; citrate, in aconcentration of about 15 mM; zinc, in a concentration of about 0.3 mM;sodium chloride, in a concentration of about 15 mM; magnesium chloride,in a concentration of about 5 mM; metacresol, in a concentration ofabout 3.15 mg/mL; and glycerol, in a concentration of about 7.6 mg/mL;and wherein the pharmaceutical composition provides for an uptake ofinsulin into the blood and/or onset of action that is between 10% and20%, between 20% and 30%, between 30% and 40% or between 40% and 50%more rapid than for compositions of rapid-acting insulin analogs whichdo not contain citrate, when measured by one or more pharmacokinetic orpharmacodynamic parameters relevant to time action, such as thosedescribed above.

In another embodiment, the pharmaceutical composition comprises insulinlispro, in a concentration of about 100 IU/mL; citrate, in aconcentration of about 25 mM; zinc, in a concentration of about 0.3 mM;sodium chloride, in a concentration of about 15 mM; magnesium chloride,in a concentration of about 5 mM; metacresol, in a concentration ofabout 3.15 mg/mL; and glycerol, in a concentration of about 4.5 mg/mL;and wherein the pharmaceutical composition provides for an uptake ofinsulin into the blood and/or onset of action that is between 10% and50% more rapid than for compositions of rapid-acting insulin analogswhich do not contain citrate, when measured by one or morepharmacokinetic or pharmacodynamic parameters relevant to time action,such as those described above.

In another embodiment, the pharmaceutical composition comprises insulinlispro, in a concentration of about 100 IU/mL; citrate, in aconcentration of about 35 mM; zinc, in a concentration of about 0.3 mM;sodium chloride, in a concentration of about 23 mM; magnesium chloride,in a concentration of about 5 mM; and metacresol, in a concentration ofabout 3.15 mg/mL; and wherein the pharmaceutical composition providesfor an uptake of insulin into the blood and/or onset of action that isbetween 10% and 50% more rapid than for compositions of rapid-actinginsulin analogs which do not contain citrate, when measured by one ormore pharmacokinetic or pharmacodynamic parameters relevant to timeaction, such as those described above.

In another embodiment, the pharmaceutical composition comprises insulinlispro, in a concentration of about 100 IU/mL; citrate, in aconcentration of about 25 mM; zinc, in a concentration of about 0.3 mM;sodium chloride, in a concentration of about 25 mM; and metacresol, in aconcentration of about 3.15 mg/mL; and wherein the pharmaceuticalcomposition provides for an uptake of insulin into the blood and/oronset of action that is between 10% and 50% more rapid than forcompositions of rapid-acting insulin analogs which do not containcitrate, when measured by one or more pharmacokinetic or pharmacodynamicparameters relevant to time action, such as those described above.FIG. 1. FIG. 1 provides a scheme depicting the amino acid sequences anddisulfide bonds of human insulin and of the rapid-acting insulin analogsthat are presently approved for use in treating meal-time excursions ofblood glucose.

When used herein, the term “composition” refers to a combination ofinsulin and the other ingredients or excipients wherein the insulin andother ingredients or excipients are combined in a single combinedformulation.

When used herein, “insulin” means human insulin or a rapid-actingstructural variant, mutant, or analog of human insulin that has thefunctional activity of but faster onset of action than human insulin.Particular rapid-acting analogs of human insulin are insulin lispro,insulin aspart, and insulin glulisine. Insulin for commercial productsmay be produced using recombinant DNA methods or by chemical synthesis.Recombinant methods are well-known and are strongly preferred. Amolecule of human insulin (CAS No. 11061-68-0) consists of two aminoacid chains, A and B, whose sequences are well-known.

The human insulin A-chain has the following sequence of amino acids:

(SEQ ID NO: 1) Gly Ile Val Glu Gln Cys Cys Thr Ser Ile Cys SerLeu Tyr Gln Leu Glu Asn Tyr Cys Asn.

The human insulin B-chain has the following sequence of amino acids:

(SEQ ID NO: 2) Phe Val Asn Gln His Leu Cys Gly Ser His Leu ValGlu Ala Leu Tyr Leu Val Cys Gly Glu Arg Gly Phe Phe Tyr Thr Pro Lys Thr.The chains are joined by two disulfide bonds: CysA7-CysB7 andCysA20-CysB19. The A-chain has an intra-chain disulfide bond atCysA6-CysA11. Human insulin has the empirical formula C₂₅₇H₃₈₃N₆₅O₇₇S₆and a molecular weight of 5808.

Insulin lispro, the drug substance in HUMALOG®, is identical to humaninsulin in terms of its primary amino acid sequence except for aninversion of the natural proline-lysine sequence on the B-chain atpositions 28 and 29 (28^(B)-L-Lysine-29^(B)-L-proline human insulin).Insulin lispro (CAS No. 133107-64-9) has been shown to be equipotent tohuman insulin on a molar basis but its effect after subcutaneousinjection is more rapid and of shorter duration than that of injectedsoluble human insulin. HUMALOG® contains m-cresol as a preservative anda stabilizer agent, a tonicity modifier (glycerol), a buffering agent(dibasic sodium phosphate), a stabilizer (zinc oxide) and pH adjustmentfor the vehicle.

A molecule of insulin lispro consists of the human insulin A-chain (SEQID NO. 1) cross-linked with the insulin lispro B-chain, whose amino acidsequence is given by SEQ ID NO:3, below:

(SEQ ID NO: 3) Phe Val Asn Gln His Leu Cys Gly Ser His Leu ValGlu Ala Leu Tyr Leu Val Cys Gly Glu Arg Gly Phe Phe Tyr Thr Lys Pro Thr.The chemical formula of insulin lispro is C₂₅₇H₃₈₃N₆₅O₇₇5₆ and itsmolecular weight is approximately 5808. One unit of insulin lispro isequivalent to 0.0347 mg insulin lispro.Insulin aspart (CAS No. 116094-23-6), the drug substance in NOVOLOG®, isanother rapid-onset insulin analog. Its structure consists of theA-chain of human insulin (SEQ ID NO. 1) and a B-chain in which the Proat B28 is replaced with Asp (Pro-B28-Asp human insulin), as reflected inthe following amino acid sequence:

(SEQ ID NO: 4) Phe Val Asn Gln His Leu Cys Gly Ser His Leu ValGlu Ala Leu Tyr Leu Val Cys Gly Glu Arg Gly Phe Phe Tyr Thr Asp Lys Thr.

Insulin aspart (28^(B) aspartic acid-human insulin) has the empiricalformula C₂₅₆H₃₈₁N₆₅O₇₉S₆ and a molecular weight of about 5826. One unitof insulin aspart corresponds to 6 nmol, corresponding with 0.035 mgsalt-free anhydrous insulin aspart.

Insulin glulisine (CAS No. 207748-29-6), the drug substance in APIDRA®,is yet another rapid-onset insulin analog. A molecule of insulinglulisine consists of human insulin A-chain (SEQ ID NO. 1) and amodified B-chain (Asn-B3-Lys, Lys-B29-Glu) compared with human insulin,as reflected in the following amino acid sequence:

(SEQ ID NO: 5) Phe Val Lys Gln His Leu Cys Gly Ser His Leu ValGlu Ala Leu Tyr Leu Val Cys Gly Glu Arg Gly Phe Phe Tyr Thr Pro Glu Thr.

Insulin glulisine (3^(B)-lysine-29^(B)-glutamic acid-human insulin) hasthe empirical formula C₂₅₈H₃₈₄N₆₄O₇₈S₆ and a molecular weight of 5823.One unit of insulin glulisine corresponds approximately to 0.0349 mg ofinsulin glulisine.

In certain embodiments, the compositions of the present invention haveconcentrations of insulin from about 100 to about 500 IU/mL. In certainembodiments, the compositions of the present invention haveconcentrations of insulin from about 100 to about 300 IU/mL. In certainembodiments, the compositions of the present invention haveconcentrations of insulin from about 100 to about 200 IU/mL. Certaincompositions comprise about 100 IU/mL. Certain compositions compriseabout 200 IU/mL. The improvements in the time action profile of theabove-referenced insulin analogs demonstrated in the present inventionare achieved through the use of certain specific concentrations ofcitrate and without the need for any additional chelating agent, such asthose described in US2012/0178675 and US2014/0113856 (e.g., EDTA), anyadditional vasodilatory agent, such as those described in US2015/0065423(e.g., nitroglycerin), or any other oligosaccharides, such as thosedescribed in US2013/0231281.

The citrate ion has the chemical name2-hydroxypropane-1,2,3-tricarboxylate, molecular formula C₆H₅O₇ ⁻³, andmolecular weight of 189. The citrate ion is widely distributed in plantsand animals and is a naturally occurring component of the diet. It is acommon metabolite in oxidative metabolism and an important component ofbone. A number of citrates are GRAS (generally regarded as safe) by theU.S. Food and Drug Administration for use in foods, including thefollowing:

GRAS Substance Formula (m.w.) CAS No. 21 CFR Citric acid C₆H₈O₇ (192.12)77-92-9 184.1033 Sodium citrate C₆H₅Na₃O₇ (258.07) 68-04-2 184.1751Potassium citrate C₆H₅O₇K₃ (324.41) 6100-05-6 184.1625 monohydrate

Various citrate-containing compounds are also included as ingredients inparenteral drug products according to the U.S. Food and DrugAdministration Inactive Ingredients database, including for example,citric acid, citric acid monohydrate, citric acid anhydrous, sodiumcitrate, anhydrous trisodium citrate, trisodium citrate dihydrate. Theparticular citrate compound used in the compositions of the presentinvention may be the acidic form or various salt forms, especially thealkali (e.g., sodium and potassium) salts and/or mono or dihydratesthereof. Of these, sodium citrate is preferred. In some embodiments, theconcentration of citrate in the compositions of the present inventionranges from about 10 to about 35 mM or about 15 mM to about 35. Inpreferred embodiments, the concentration of citrate ranges from about 10to about 30 mM or about 15 to about 30 mM. In certain preferredembodiments, the concentration of citrate ranges from about 15 to about25 mM. In certain embodiments, the concentration of citrate ranges fromabout 15 to about 25 mM. In certain embodiments, the concentration ofcitrate ranges from about 15 to about 20 or about 20 to about 25 mM.Certain compositions have citrate concentrations of about 15, about 20,about 25, about 30 or 35 mM.

Although the addition of citrate has been found to result inimprovements in time action, the addition of citrate also leads togreater liabilities from a stability standpoint. Thus, in order to havesufficient chemical and physical stability for long-term storage anduse, the compositions of the present invention further comprise containstabilizing agents, such as zinc, magnesium, chloride and surfactant(s).

Zinc oxide may be added to provide the desired stoichiometry of zincions. Insulin hexamers have 2 specific, high affinity zinc bindingsites. Zinc ions incorporated into such hexamers are sometimes referredto as “bound” zinc. Currently available zinc-containing formulationsinclude between about 2 and 4 zinc ions per hexamer of insulin. Somecommercial insulin compositions have about 2.4 ions of zinc per sixmolecules of insulin (HUMULIN® R U-500), and some have about 3.0 ions ofzinc per six molecules of insulin (HUMALOG®, NOVOLOG®). The 100 U/mLformulations of insulin lispro (HUMALOG®) and insulin aspart (NOVOLOG®)have about 3.0 ions of zinc per six molecules of insulin, whichcorresponds with a concentration of about 0.3 mM. The currentlyavailable 200 U/mL formulation of HUMALOG® has about 3.5 ions of zincper six molecules of insulin, which corresponds with a zincconcentration of about 0.7 mM. The currently available 100 U/mLformulation of human insulin sold by Eli Lilly and Company (HUMULIN® R)contains about 2.3 ions of zinc per six molecules of insulin, whichcorresponds with a zinc concentration of about 0.23 mM.

The compositions of the present invention have a zinc concentrationsufficient to provide at least enough zinc ions for the insulinmolecules to form stabilizing hexamers. Thus, the compositions of thepresent invention must include sufficient zinc to provide at least 2ions of zinc per hexamer of insulin. The compositions of certainembodiments of the present invention have a zinc concentration fromabout 0.2 mM to about 0.8 mM. When the insulin concentration is 100IU/mL, the zinc concentration in certain embodiments of the presentinvention is about 0.3 mM (about 3.0 Zn ions/six insulin molecules). Incertain embodiments of the present invention having, for example,insulin concentrations of about 100 U/mL, about 200 U/mL, about 300 U/mLor about 500 U/mL, the minimum zinc concentration necessary to provide 2ions of zinc per insulin hexamer would be about 0.2 mM, about 0.4 mM,about 0.6 mM or about 1 mM, respectively.

The inclusion of excess zinc—i.e., more zinc than would be bound ininsulin hexamers—may be used to further stabilize compositions of thepresent invention. Such zinc is sometimes referred to as “free” or“unbound” zinc. In certain compositions of the present invention, theinclusion of excess free or unbound zinc has been found to have astabilizing effect. Compositions having 100 about U/mL of insulin lisproand zinc concentrations up to about 1 mM—which would constitute about0.2 mM bound and about 0.8 mM unbound or free zinc—have been found to beboth fast acting and stable. The inclusion of too much free or unboundzinc, however, may attenuate the improvements in time action. Forexample, a composition having about 100 U/mL of insulin lispro with azinc concentration of about 5 mM—which would constitute about 4.8 mMunbound zinc—was found to not have the improvements in time action seenin compositions with lower zinc concentrations. In certain embodimentsof the present invention, the concentration of zinc ranges from about0.2 to about 2 mM, about 0.4 to about 1 mM, or about 0.6 to about 0.9mM. In certain embodiments, the concentration of zinc is about 0.6,about 0.7, about 0.8 or about 0.9 mM.

The inclusion of magnesium (Mg⁺²) in compositions of the presentinvention has also been found to have a stabilizing effect. Magnesiumions may be provided in a variety of manners, such as through theaddition of magnesium chloride, which has a molecular formula of MgCl₂and molecular weight of 95.211.

While magnesium may have stabilizing effects in certain compositions,concentrations which exceed the concentration of citrate will result ininsulin precipitation. Thus, the maximum amount of magnesium that may beincluded is limited by the amount of citrate that is included. Forexample, when MgCl₂ is used a stabilizing agent in the compositions ofthe present invention, the molar ratio of magnesium to citrate rangesfrom about 1:10 to about 1:2. Preferably the molar ratio of magnesiumchloride to citrate ranges from about 1:5 to about 1:3. Thus, forexample, in order to achieve a molar ratio of magnesium to citrate fromabout 1:10 to about 1:2 in a composition wherein the citrateconcentration is between about 10 to about 30 mM, the concentration ofmagnesium would be between about 1 and about 15 mM. Similarly, in orderto achieve a molar ratio of magnesium to citrate of about 1:5 to about1:3 in a composition wherein the citrate concentration is 25 mM, theconcentration of magnesium would range from 5 to 8.3 mM. In certainembodiments, the concentration of magnesium ranges from about 1 about 15mM. In certain embodiments, the concentration of magnesium ranges fromabout 1 about 5, about 5 to about 10 or about 10 to about 15 mM. Theconcentration of magnesium chloride in certain embodiments of thepresent invention ranges from 1 to about 5 mM (˜0.48 mg/mL). In certainembodiments, the concentration of magnesium is about 5 mM.

The inclusion of certain concentrations of chloride ions (Cl⁻) incompositions of the present invention has also been found to have astabilizing effect. Chloride ions may be provided in a variety ofmanners, including through the use of MgCl₂ to provide magnesium, asdescribed above, or through the addition of sodium chloride. Thecompositions of certain embodiments of the present invention comprisesodium chloride. The molecular formula of sodium chloride is NaCl andits molecular weight is 58.44. Sodium chloride is used in some currentlyavailable formulations of rapid acting insulin analogs, such as APIDRA®(insulin glulisine), which comprises 5 mg/mL sodium chloride, NOVOLOG®(insulin aspart), which comprises 0.58 mg/mL sodium chloride. While theaddition of certain quantities of chloride, e.g., through the inclusionin the composition of chloride-containing excipients such as MgCl₂ orNaCl, has been found to have a stabilizing effect, if the total chloridecontent of the composition is too high, the insulin in the compositionmay crystallize at low temperatures. Thus, the total chloride content ofthe composition must be taken into consideration.

In order to determine the total chloride content of a composition, onemust take into consideration not just chloride ions which may be addedto the composition through the addition of magnesium chloride and/orsodium chloride as stabilizing agents, but also through the addition ofother components, for example with the insulin bulk activepharmaceutical ingredient (API), through the addition of small amountsof HCl which may be necessary for pH adjustments, and/or in connectionwith the provision of Zn, which may be added in the form of a solutionprepared by solubilizing zinc oxide (ZnO) with HCl.

In certain embodiments, sodium chloride is used to provide the amount ofadditional chloride needed to reach the target chloride concentration orconcentration range—i.e., the amount of sodium chloride to be added isdetermined by subtracting from the target chloride concentration theamount of chloride provided through the addition of other components,such as through the addition of insulin API, magnesium chloride and/orany HCl which might be necessary for pH adjustments and/orsolubilization of zinc oxide. For example, if the target chlorideconcentration in a formulation is about 20 mM, and 15 mM of chloride isprovided through a combination of the bulk insulin API, magnesiumchloride, and HCl used for pH adjustments, 5 mM of sodium chloride mustbe added. Persons of skill in the art will understand that the quantityof sodium chloride to be added in such formulations may be determinedeither by: (1) prospectively calculating the amount of additionalchloride that will be needed based on the theoretical chloride contentadded through other sources; or (2) preparing a formulation of all ofthe excipients except for sodium chloride, measuring the chloridecontent of that formulation, and calculating the difference between thatamount and the targeted chloride concentration or concentration range.The chloride content of an aqueous formulation maybe measured using avariety of known techniques, such as by titration or ion-selectiveelectrode methods.

In addition, the low temperature crystallization issues associated withrelatively high chloride concentrations have also been found to besensitive to citrate concentrations. Thus, compositions of the presentinvention having citrate concentrations at the lower end of the rangeprovided for herein may be more tolerant of relatively higher chlorideconcentrations than compositions having citrate concentrations at thehigher end of the range provided for herein. For example, the additionof sodium chloride concentrations as high as 50-75 mM to formulationscontaining 25 mM citrate have been observed to lead to low temperaturecrystallization issues, but such issues are not consistently observedeither when 50 mM sodium chloride is added to a 15 mM citrateformulation or when 25 mM sodium chloride is added to a 25 mM citrateformulation.

For the sake of clarity, when used herein, the terms “chloride” or“total chloride” refer to the total amount of chloride ions in acomposition provided in connection with the addition of any component,e.g., the source(s) of chloride ions in a composition which is stated tocomprise 25 mM chloride or total chloride include any chloride providedthrough the addition of MgCl₂, NaCl and/or any HCl needed for pHadjustments or solubilization of ZnO. On the other hand, the terms“magnesium chloride,” “MgCl₂,” “sodium chloride” and “NaCl” refer to theamount of these particular salts that are added to a composition. Thus,in a composition which is described as comprising 5 mM magnesiumchloride and 10 mM sodium chloride, for example, the concentration ofchloride, or total chloride, includes the combined amount of chlorideions provided by the magnesium chloride, sodium chloride and any othersource, such as HCl.

In certain embodiments of the present invention, the total chlorideconcentration ranges from about 10 to about 60 mM. In certainembodiments, the total chloride concentration ranges from about 15 toabout 35 mM. In certain embodiments, the total chloride concentrationranges from about 20 to about 25 mM. In certain embodiments, the totalchloride concentration is about 20 mM, about 21 mM, about 22 mM, about23 mM, about 24 mM or about 25 mM.

In certain embodiments of the present invention, the compositioncomprises sodium chloride in a concentration ranging from about 1 toabout 50 mM. In certain embodiments, the concentration of sodiumchloride is from about 1 to about 25 mM. In certain embodiments, theconcentration of sodium chloride ranges from about 1 to about 20 mM. Theconcentration of sodium chloride in certain embodiments of the presentinvention ranges from about 15 mM (˜0.88 mg/mL) to about 25 mM (˜2.0mg/mL). In certain embodiments, the concentration of sodium chloride isabout 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8,about 9, about 10, about 11, about 12, about 13, about 14, about 15,about 16, about 17, about 18, about 19 or about 20 mM.

If necessary to achieve sufficient chemical and physical stability, thecomposition may further comprise one or more additional stabilizingagents. Exemplary stabilizing agents include surfactants. It hassurprisingly been discovered that combinations of surfactant andmagnesium as stabilizing agents may have greater-than-additive orsynergistic stabilizing effects in compositions of the presentinvention.

Examples of surfactants disclosed for use in parenteral pharmaceuticalcompositions include polysorbates, such as polysorbate 20 (TWEEN® 20),polyethylene glycols such as PEG 400, PEG 3000, TRITON™ X-100,polyethylene glycols such as polyoxyethylene (23) lauryl ether (CASNumber: 9002-92-0, sold under trade name BRIJ®), alkoxylated fattyacids, such as MYRJ™, polypropylene glycols, block copolymers such aspoloxamer 188 (CAS Number 9003-11-6, sold under trade name PLURONIC®F-68) and poloxamer 407 (PLURONIC® F127), sorbitan alkyl esters (e.g.,SPAN®), polyethoxylated castor oil (e.g., KOLLIPHOR®, CREMOPHOR®) andtrehalose and derivatives thereof, such as trehalose laurate ester. Incertain embodiments, the surfactant is selected from the groupconsisting of polyoxyethylene (23) lauryl ether, poloxamer 188 andtrehalose laurate ester. A preferred surfactant is poloxamer 188. Incertain embodiments, the concentration of surfactant, such as poloxamer188, ranges from about 0.001 to about 2% w/v, about 0.001 to about 0.2%w/v, about 0.03 to about 0.12% w/v, or about 0.06 to about 0.09% w/v. Incertain embodiments, the concentration of poloxamer 188 is about 0.06,about 0.07, about 0.08 or about 0.09% w/v.

In addition, the compositions of the present invention include one ormore preservatives, which provide anti-microbial properties and mayfurther provide stability benefits. The compositions are sterile whenfirst produced, however, when the composition is provided in a multi-usevial or cartridge, an anti-microbial preservative compound or mixture ofcompounds that is compatible with the other components of theformulation is typically added at sufficient strength to meet regulatoryand pharmacopoeial anti-microbial preservative requirements. See U.S.Pharmacopeia Monographs. Insulin lispro injection. USP29-NF24; BritishPharmacopeia Monographs 2008 Volume III: Insulin aspart injection; U.S.Pharmacopeia Monographs. Insulin assays; and U.S. Pharmacopeia generalchapters. USP29-NF24. Rockville, Md,: U.S. Pharmacopeial Convention;2005. Antimicrobial effectiveness testing; pp. 2499-2500. Preferredpreservatives are aryl acids and phenolic compounds, or mixtures of suchcompounds.

The compositions of the present invention thus include one or morepreservatives. Effective concentrations can be ascertained readily usingthe methods referenced above. Preservatives commonly used in insulinproducts include phenol (CAS No. 108-95-2, molecular formula C₆H₅OH,molecular weight 94.11,), and m-cresol (CAS No. 108-39-4, molecularformula C₇H₈O, molecular weight 108.14). Present commercialcompositions, for example, contain 3.15 mg/mL m-cresol (HUMALOG® andAPIDRA®), 1.72 mg/mL m-cresol and 1.50 mg/mL phenol (NOVOLOG®), and 2.5mg/mL m-cresol (HUMULIN® R U-500). The compositions of the presentinvention include one or more preservatives. In an embodiment thepreservative is selected from the group consisting of phenol andmetacresol and mixtures thereof. Preferably the preservative ismetacresol. In certain embodiments the metacresol concentration is fromabout 2.5 mg/mL to about 3.8 mg/mL. Preferably the concentration ofmetacresol is about 3.15 mg/mL.

It is desirable to approximately match the tonicity (i.e., osmolality)of body fluids at the injection site as closely as possible whenadministering the compositions because solutions that are notapproximately isotonic with body fluids can produce a painful stingingsensation when administered. Thus, it is desirable that the compositionsbe approximately isotonic with body fluids at the sites of injection. Ifthe osmolality of a composition in the absence of a tonicity agent issufficiently less than the osmolality of the tissue (for blood, about300 mOsmol/kg; the European Pharmacopeial requirement for osmolalityis >240 mOsmol/kg), then a tonicity agent should generally be added toraise the tonicity of the composition to about 300 mOsmol/kg. Typicaltonicity agents are glycerol (glycerin) and sodium chloride. If theaddition of a tonicity agent is required, glycerol is preferred. Theamount of tonicity agent to add is readily determined using standardtechniques. Remington: The Science and Practice of Pharmacy, David B.Troy and Paul Beringer, eds., Lippincott Williams & Wilkins, 2006, pp.257-259; Remington: Essentials of Pharmaceutics, Linda Ed Felton,Pharmaceutical Press, 2013, pp. 277-300. In certain embodiments theconcentration of glycerol is from about 1 to about 16 mg/mL.

In certain embodiments, the pharmaceutical composition is stable understorage and use conditions. When used herein, the term “stable” refersto both chemical and physical stability, as indicated, for example, by aloss of insulin potency of less than 5%, A-21 desamido formation of notmore than 1.5%, development of substances other than insulin and A-21desamido (collectively referred to as other related substances (ORS)) ofnot more than 4%, high molecular weight (HMW) formation of less than1.5%, and the maintenance of a clear and colorless solution with noprecipitate. Such properties may be measured by known techniques,including for example, those summarized in the studies described below.In certain embodiments, the pharmaceutical composition is stable toallow for storage of at least 24 months at 2-8° C. In certainembodiments, the pharmaceutical composition is stable to allow for up to28 days in-use at temperatures of up to 30° C. for vials or cartridgesin re-usable pen injectors. In certain embodiments, the pharmaceuticalcomposition is stable to allow for storage of at least 36 months at 2-8°C. In certain embodiments, the pharmaceutical composition is stable toallow for up to 32 days in-use at temperatures of up to 30° C. for vialsor cartridges in re-usable pen injectors. In certain embodiments, thecomposition is stable to allow for use in a pump device for continuoussubcutaneous insulin infusion therapy for up to 7 days.

Citrate, which as noted above is added to contribute to improvements intime action, is also known to also have buffering properties at certainpH levels, but if desired an additional buffering compound may beincluded. Examples of such buffering compounds are phosphate buffers,such as dibasic sodium phosphate, or sodium acetate andtris(hydroxymethyl)aminomethane (TRIS). Phosphate or TRIS buffers arepreferred. The pH for commercial insulin compositions is usually in therange of 7.2 to 7.6, with 7.4±0.1 as a common target pH. The pH ofcompositions of the present invention is typically 7.0 to 7.8 and it isadjusted using physiologically appropriate acids and bases, typicallyhydrochloric acid 10% and sodium hydroxide 10%. Preferably, the pH isabout 7.4.

The route of administration for the compositions of the presentinvention will typically be by self-administered subcutaneous injection,e.g., by use of a syringe or a pen device, or by continuous subcutaneousinsulin infusion therapy with an insulin pump device, thoughintravenous, intradermal, or intraperitoneal routes may also be used.Preferably, the route of administration is by self-administeredsubcutaneous injection. The dose of active agent injected will bedetermined by the patient in consultation with the patient's physician.

Additional embodiments of the present invention include those describedbelow:

A pharmaceutical composition comprising: an insulin; citrate; zinc;magnesium; chloride; a surfactant; and a preservative.

The pharmaceutical composition of any of the above-describedembodiments, wherein the insulin is selected from the group consistingof human insulin, insulin lispro, insulin aspart or insulin glulisine.

The pharmaceutical composition of any of the above-describedembodiments, wherein the insulin is insulin lispro.

The pharmaceutical composition of any of the above-describedembodiments, wherein the insulin concentration is from about 100 toabout 500 U/mL.

The pharmaceutical composition of any of the above-describedembodiments, wherein the insulin concentration is from about 100 toabout 300.

The pharmaceutical composition of any of the above-describedembodiments, wherein the insulin concentration is from about 100 toabout 200 U/mL.

The pharmaceutical composition of any of the above-describedembodiments, wherein the insulin concentration is about 100 U/mL. Thepharmaceutical composition of any of the above-described embodiments,wherein the insulin concentration is about 100 U/mL.

The pharmaceutical composition of any of the above-describedembodiments, wherein the concentration of citrate is from about 10 toabout 35 mM.

The pharmaceutical composition of any of the above-describedembodiments, wherein the concentration of citrate is from about 10 toabout 30 mM.

The pharmaceutical composition of any of the above-describedembodiments, wherein the concentration of citrate is from about 15 toabout 35 mM.

The pharmaceutical composition of any of the above-describedembodiments, wherein the concentration of citrate is from about 15 toabout 30 mM.

The pharmaceutical composition of any of the above-describedembodiments, wherein the concentration of citrate is from about 15 toabout 25 mM.

The pharmaceutical composition of any of the above-describedembodiments, wherein the concentration of citrate is from about 15 toabout 20 mM.

The pharmaceutical composition of any of the above-describedembodiments, wherein the concentration of citrate is from about 20 toabout 25 mM.

The pharmaceutical composition of any of the above-describedembodiments, wherein the concentration of zinc is from about 0.2 toabout 2 mM.

The pharmaceutical composition of any of the above-describedembodiments, wherein the concentration of zinc is from about 0.3 toabout 1 mM.

The pharmaceutical composition of any of the above-describedembodiments, wherein the concentration of zinc is from about 0.4 toabout 1 mM.

The pharmaceutical composition of any of the above-describedembodiments, wherein the concentration of zinc is from about 0.6 toabout 0.9 mM.

The pharmaceutical composition of any of the above-describedembodiments, wherein the molar ratio of magnesium to citrate is fromabout 1:10 to about 1:2.

The pharmaceutical composition of any of the above-describedembodiments, wherein the molar ratio of magnesium to citrate is fromabout 1:5 to about 1:3.

The pharmaceutical composition of any of the above-describedembodiments, wherein the concentration of magnesium is from about 1 toabout 15 mM.

The pharmaceutical composition of any of the above-describedembodiments, wherein the concentration of magnesium is from about 1 toabout 5 mM.

The pharmaceutical composition of any of the above-describedembodiments, wherein the concentration of magnesium is from about 5 toabout 10 mM.

The pharmaceutical composition of any of the above-describedembodiments, wherein the concentration of magnesium is from about 10 toabout 15 mM.

The pharmaceutical composition of any of the above-describedembodiments, wherein the concentration of magnesium is from about 3 toabout 8 mM.

The pharmaceutical composition of any of the above-describedembodiments, wherein the magnesium concentration is provided through theinclusion of magnesium chloride.

The pharmaceutical composition of any of the above-describedembodiments, wherein the total chloride concentration is from about 10to about 60 mM.

The pharmaceutical composition of any of the above-describedembodiments, wherein the total chloride concentration is from about 15to about 35 mM.

The pharmaceutical composition of any of the above-describedembodiments, wherein the total chloride concentration is from about 15to about 30 mM.

The pharmaceutical composition of any of the above-describedembodiments, wherein the total chloride concentration is from about 20to about 25 mM.

The pharmaceutical composition of any of the above-describedembodiments, wherein the composition comprises sodium chloride in aconcentration ranging from about 1 to about 50 mM.

The pharmaceutical composition of any of the above-describedembodiments, wherein the composition comprises sodium chloride in aconcentration ranging from about 1 to about 25 mM.

The pharmaceutical composition of any of the above-describedembodiments, wherein the composition comprises sodium chloride in aconcentration ranging from about 1 to about 20 mM.

The pharmaceutical composition of any of the above-describedembodiments, wherein the concentration of surfactant is from about 0.001to about 0.2% w/v.

The pharmaceutical composition of any of the above-describedembodiments, wherein the surfactant is poloxamer 188.

The pharmaceutical composition of any of the above-describedembodiments, wherein the concentration of poloxamer 188 is from about0.003 to about 0.2% w/v.

The pharmaceutical composition of any of the above-describedembodiments, wherein the concentration of poloxamer 188 is from about0.03 to about 0.12% w/v.

The pharmaceutical composition of any of the above-describedembodiments, wherein concentration of poloxamer 188 is from about 0.06to about 0.09% w/v.

The pharmaceutical composition of any of the above-describedembodiments, wherein the preservative is selected from the groupconsisting of phenol and metacresol.

The pharmaceutical composition of any of the above-describedembodiments, wherein the preservative is metacresol.

The pharmaceutical composition of any of the above-describedembodiments, wherein the metacresol concentration is from about 2.5 toabout 3.8 mg/mL.

The pharmaceutical composition of any of the above-describedembodiments, further comprising a tonicity agent.

The pharmaceutical composition of any of the above-describedembodiments, comprising glycerol as a tonicity agent.

The pharmaceutical composition of any of the above-describedembodiments, comprising glycerol as a tonicity agent in a concentrationfrom about 1 to about 16 mg/mL.

The pharmaceutical composition of any of the above-describedembodiments, further comprising an additional buffer.

The pharmaceutical composition of any of the above-describedembodiments, wherein the pH of the composition is from about 7.0 toabout 7.8.

The pharmaceutical composition of any of the above-describedembodiments, wherein the composition provides for an uptake of insulininto the blood that is at least 20% more rapid than for compositionswhich contain the same insulin but which do not contain citrate.

The pharmaceutical composition of any of the above-describedembodiments, wherein the composition provides for an onset of actionthat is at least 20% more rapid than for compositions which contain thesame insulin but which do not contain citrate.

The pharmaceutical composition of any of the above-describedembodiments, wherein the composition provides for an uptake of insulininto the blood that is at least 30% more rapid than for compositionswhich contain the same insulin but which do not contain citrate.

The pharmaceutical composition of any of the above-describedembodiments, wherein the composition provides for an onset of actionthat is at least 30% more rapid than for compositions which contain thesame insulin but which do not contain citrate.

The pharmaceutical composition of any of the above-describedembodiments, wherein the composition is stable to allow for storage ofat least 24 months at 2-8° C.

The pharmaceutical composition of any of the above-describedembodiments, wherein the composition is stable to allow for up to 28days in-use at temperatures of up to 30° C. for vials or cartridges inre-usable pen injectors.

The pharmaceutical composition of any of the above-describedembodiments, wherein the composition is stable to allow for storage ofat least 36 months at 2-8° C.

The pharmaceutical composition of any of the above-describedembodiments, wherein the composition is stable to allow for up to 32days in-use at temperatures of up to 30° C. for vials or cartridges inre-usable pen injectors.

The pharmaceutical composition of any of the above-describedembodiments, wherein the composition is stable to allow for use in apump device for continuous subcutaneous insulin infusion therapy for upto 7 days.

The pharmaceutical composition of any of the above-describedembodiments, wherein the composition does not include any additionalchelating agent.

The pharmaceutical composition of any of the above-describedembodiments, wherein the composition does not include EDTA.

The pharmaceutical composition of any of the above-describedembodiments, wherein the composition does not include EDTA, ethyleneglycol tetraacetic acid (EGTA), alginic acid, alpha lipoic acid,dimercaptosuccinic acid (DMSA), or 1,2-diaminocyclohexanetetraaceticacid (CDTA).

The pharmaceutical composition of any of the above-describedembodiments, wherein the composition does not include any vasodilatoryagent.

The pharmaceutical composition of any of the above-describedembodiments, wherein the composition does not include any nitroglycerin.

The pharmaceutical composition of any of the above-describedembodiments, wherein the composition does not include adenosine,endothelium-derived hyperpolarizing factor, a phosphodiesterase type 5(PDES) inhibitor, a potassium channel opener, prostacyclin, forskolin,nitroglycerin, a nitric oxide forming agent, amyl nitrite, ornitroprusside.

The pharmaceutical composition of any of the above-describedembodiments, wherein the composition does not include anyoligosaccharides.

A method of treating diabetes comprising administering to a human inneed thereof an effective dose of the pharmaceutical composition of anyof the above-described embodiments.

The pharmaceutical composition of any of the above-described embodimentsfor use in therapy.

The pharmaceutical composition of any of the above-described embodimentsfor use in the treatment of diabetes.

The pharmaceutical composition of any of the above-described embodimentsfor use in the manufacture of a medicament for the treatment ofdiabetes.

A multi-use vial comprising any one of the pharmaceutical compositionsof any of the above-described embodiments.

An re-usable pen injector comprising any one of the pharmaceuticalcompositions of any of the above-described embodiments.

A pump device for continuous subcutaneous insulin infusion therapycomprising any one of the pharmaceutical compositions of any of theabove-described embodiments.

The invention is further illustrated by the following examples, whichare not to be construed as limiting.

EXAMPLES Pharmacokinetic and Pharmacodynamic Studies

Insulin Lispro Formulated with 35 mM Citrate, 0.3 mM Zinc, 5 mM MgCl₂and 23 mM NaCl

Diabetic (Alloxan induced), castrated, male Yucatan miniature swine withpreviously fitted vascular access ports were used under the supervisionof staff and veterinarians. The diabetic animals are housed individuallyand have access to fresh water at all times. They are fed two meals perday of a standard diet and receive appropriate maintenance basal andprandial insulin twice per day to manage their diabetic condition.

Test article (Composition A in the table below) was formulated andshipped to the test site and Humalog® insulin control was a commercialvial on site at test site:

TABLE 1 Name Formulation Composition Composition A 108 U/mL insulinlispro 35 mM citrate 5 mM MgCl₂ 23 mM NaCl 0.3 mM zinc 3.15 mg/mLm-cresol pH 7.4 Humalog ® 100 U/mL KPB 1.88 mg/mL dibasic sodiumphosphate 16 mg/mL glycerol 3.15 mg/mL meta-cresol 0.3 mM zinc pH 7.4

The day prior to study, animals were fed half their daily ration andreceived 0.2 U/kg Humalog® Mix 75/25 Insulin at their morningmaintenance administration. All study animals were food-fasted overnightand did not receive their evening insulin or meal prior to drugadministration on study day.

On the morning of study, all animals were placed into slings forrestraint and had their vascular access ports accessed (equipped forblood sampling) and checked for patency. The animals were randomlyplaced into treatment groups. This was a full crossover design withn=13. One animal did not participate in the Composition A treatmentgroup yielding n=12 for that treatment group.

After two baseline blood samples were collected (−30 and −20 min), theanimals were returned to their pens and were fed ˜300 g. Twenty minutesafter the presentation of the fully consumed meal, the animals wereinjected with test article subcutaneously in the flank (0 min) with aTerumo insulin syringe (0.3 or 0.5 ml with ½″ needle). All study animalshad access to clean, fresh water throughout the remaining bloodcollection period.

Serial blood samples (2.0 mL each) were collected from each animal atthe following time points: −30, −20 (then immediately Fed), 0 (justbefore dose), 5, 10, 15, 30, 45, 60, 75, 90, 105, 120, 150, 180, 240,and 360 minutes following the SC dosing.

Blood samples (anticoagulant: none [serum]) were maintained at ambienttemperature for at least 30 minutes but no more than 2 hours to allowfor clotting. Serum was then separated by centrifugation and dividedinto two aliquots and stored frozen at approximately −70° C.

Serum glucose concentrations were determined using an automated AU480Clinical Chemistry Analyzer (Beckman Coulter, Inc., Brea, Calif.).Aliquot for PK was shipped to EMD Millipore Corp., St. Charles, Mo. ondry ice by a next day shipping service and included a detailed samplemanifest.

Serum glucose data are represented in Table 2 below as Mean(mg/dL)+/−SEM unless otherwise specified.

TABLE 2 Humalog ® Composition A Time (min) AVG SEM AVG SEM −30 308.513.2 294.6 10.0 −20 311.8 13.3 296.0 10.3 0 321.8 13.3 311.4 11.7 5325.8 14.5 319.6 9.6 10 320.2 13.0 282.3 11.2 15 297.5 21.7 243.8 11.630 249.1 13.3 168.3 14.5 45 191.9 19.1 125.7 15.2 60 148.2 17.9 95.814.2 75 113.5 15.4 75.8 13.3 90 89.8 14.2 61.2 12.0 105 68.7 11.1 53.711.0 120 53.5 9.4 44.8 10.5 150 46.5 8.0 44.9 10.2 180 44.8 9.6 43.110.5 240 74.5 17.1 62.8 11.2 360 109.7 27.0 103.9 21.8

To statistically compare the change of serum glucose under differentformulations at different time points, derivative analysis was utilized.The change of blood glucose at each time point is characterized by thefirst order derivative (instant directional change) at current timepoint. Polynomials of up to order 10 is fitted to the time course datafor each individual animal. The optimal order of the polynomial isselected using Bayesian information Criterion (BIC). The rate of changeat each time point is calculated as the derivative of the fittedpolynomial curve at that time point. Once the derivatives are obtained,ANOVA model is fitted to compare the different formulations at eachtime. Animal to animal variation is accounted for in the ANOVA model.The contrasts are constructed to compare the different formulations andthe p-values are adjusted using R package “multcomp.” Animals are dosedafter time 0; therefore, time 0 is not included in the analysis. Fortime points beyond 150 minutes, there are not enough time points torobustly estimate the derivative, therefore, these time points are alsoexcluded in the statistical analysis.

Composition A resulted in a significantly (p≤0.01) faster decrease inserum glucose at 5, 10, and 15 minutes post dose compared to Humalog®.

Insulin levels for serum PK samples for the Composition A and Humalog®treatment groups are measured using a total insulin RIA. Lower and upperlimits of quantitation for the assay are 20 pM and 5000 pM,respectively. Values below the lower limit of quantitation are assumedto be 20 pM. Non-compartmental pharmacokinetic analyses are performedusing Phoenix WinNonlin v6.3.

TABLE 3 Tmax Cmax AUC_(INF) CL/F Name (min) (nM) (min*nM) (mL/min/kg)Humalog ® Mean 53.1 1.37 157 8.70 N = 13 SE 7.52 0.246 16.9 0.871 Median45.0 1.04 127 9.48 Composition A Mean 23.8 1.48 152 8.46 N = 12 SE 6.070.130 10.5 0.822 Median 12.5 1.35 154 7.80 Abbreviations: Tmax—time tomaximal concentration, Cmax—maximum concentrations, AUCINF—area underthe curve from 0 to infinity, CL/F—Clearance/bioavailability Mean andmedian Tmax are 55% and 72% earlier in Composition A, respectively, thanwith Humalog.Insulin Lispro Formulated with 25-35 mM Citrate, 0.285 mM Zinc, 5 mMMgCl₂ and 15-23 mM NaCl.

A study of compositions comprising varying concentrations of citrate andzinc, magnesium chloride and sodium chloride is performed in diabetic(Alloxan induced), castrated, male Yucatan miniature swine followinggenerally the procedures described above.

Test articles (Compositions A′ and B in the table below) are formulatedand shipped to the test site, and Humalog® insulin control is acommercial vial on site at test site:

TABLE 4 Name Formulation Composition Composition A′ 95 U/mL insulinlispro 35 mM citrate 5 mM MgCl₂ 23 mM NaCl 0.285 mM zinc 2.99 mg/mLm-cresol 1.79 mg/mL dibasic sodium phosphate 15.2 mg/mL glycerol pH 7.4Composition B 95 U/mL insulin lispro 25 mM citrate 5 mM MgCl₂ 15 mM NaCl0.285 mM zinc 2.99 mg/mL m-cresol 1.79 mg/mL dibasic sodium phosphate15.2 mg/mL glycerol pH 7.4 Humalog ® 100 U/mL KPB 1.88 mg/mL dibasicsodium phosphate 16 mg/mL glycerol 3.15 mg/mL meta-cresol 0.3 mM zinc pH7.4

Study is designed as a 21 pig full cross-over design to study all pigson all treatments (n=21). One animal is excluded from the Humalog®treatment group for baseline blood glucose <200 mg/dl, and one animal isexcluded from the Humalog® treatment group for a non-patent port, son=19 for that treatment group. One animal is excluded from theComposition A′ treatment group for a reported miss dose at time ofinjection, so n=20 for that treatment group. One animal is excluded fromthe Composition B treatment group for baseline blood glucose <200 mg/dl,so n=20 for that treatment group.

Serum glucose data are presented in Table 5 below as mean (mg/dL)+/−SEM.

TABLE 5 Time Humalog ® Composition A′ Composition B (min) AVG SEM AVGSEM AVG SEM −30 291.1 8.9 288.3 8.7 284.1 11.4 −20 296.8 8.7 296.5 9.3289.0 12.6 0 311.5 10.6 311.1 10.5 297.5 13.8 5 320.3 8.9 319.9 10.6296.8 15.2 10 319.5 9.8 299.2 12.6 280.4 14.4 15 306.3 11.8 265.0 14.3248.7 16.5 30 262.8 17.1 206.8 21.9 185.5 19.3 45 223.6 19.4 177.1 24.3156.0 20.8 60 195.7 19.6 162.4 24.4 140.9 20.9 75 168.3 19.2 148.3 23.6131.0 21.9 90 150.3 18.4 139.8 22.9 112.9 19.1 105 130.0 17.0 128.5 21.5107.5 18.1 120 122.8 17.2 123.2 20.9 95.4 16.5 150 89.6 12.7 107.7 19.676.5 13.2 180 70.1 10.6 98.3 18.0 66.0 11.3 240 64.2 9.9 92.8 16.3 66.09.8 360 79.0 12.4 100.3 15.2 90.3 11.0

To statistically compare the change of serum glucose under differentformulations at different time points, derivative analysis, followinggenerally the procedure described above, was utilized. Compositions A′and B each resulted in significantly (p≤0.01) faster decrease in serumglucose at 5, 10, and 15 minutes post dose compared to Humalog®.

Serum insulin concentrations and PK parameters are generated andanalyzed generally as described above, and PK results are provided intable 6 below.

TABLE 6 Tmax Cmax AUC_(INF) CL/F Compound (min) (nM) (min*nM)(mL/min/kg) Humalog ® Mean 52.9 0.791 110 12.1 N = 19 SE 6.42 0.137 10.10.906 Median 45 0.569 97.9 12.3 Composition A′ Mean 48.8 0.757 106 12.6N = 20 SE 8.11 0.125 7.53 0.991 Median 30 0.675 99.9 12.0 Composition BMean 27.3 0.885 101 13.3 N = 20 SE 4.57 0.105 8.06 1.11 Median 15 0.82796.3 12.5 Median Tmax results are 33% and 67% earlier in Compositions A′and B than Humalog ®, and mean Tmax is 48% earlier in Composition B thanHumalog.Insulin Lispro Formulated with 15 mM Citrate, 0.285 mM Zinc, 5 mM MgCl₂and 15 mM NaCl

A study on compositions comprising citrate, zinc, magnesium chloride andsodium chloride is performed in diabetic (Alloxan induced), castrated,male Yucatan miniature swine following generally the proceduresdescribed above. Composition C was formulated and shipped to the testsite, and Humalog® control is a commercial vial.

TABLE 7 Name Formulation Composition Composition C 95 U/mL insulinlispro 15 mM citrate 5 mM MgCl₂ 15 mM NaCl 0.285 mM zinc 2.99 mg/mLm-cresol 1.79 mg/mL dibasic sodium phosphate 15.2 mg/mL glycerol pH 7.4Humalog ® 100 U/mL KPB 1.88 mg/mL dibasic sodium phosphate 16 mg/mLglycerol 3.15 mg/mL meta-cresol 0.3 mM zinc pH 7.4

Study is designed as a 13 pig full cross-over design to study all pigson all treatments (n=13). One animal is excluded from the Composition Ctreatment group for baseline blood glucose <200 mg/dl, and one animal isexcluded from the Composition C treatment group for a reported miss doseat time of injection, resulting in n=11 for that treatment group.

Serum glucose data are presented in Table 8 below as mean (mg/dL)+/−SEM.

TABLE 8 Humalog ® Composition C Time (min) AVG SEM AVG SEM −30 292.2 9.4297.8 10.6 −20 302.6 9.1 306.9 11.0 0 316.8 12.0 324.4 10.7 5 319.9 12.3330.8 12.8 10 312.9 13.2 312.0 11.2 15 311.4 14.8 288.4 15.1 30 248.722.2 225.5 15.0 45 203.5 22.9 189.1 18.7 60 162.8 23.9 152.2 19.9 75135.6 24.7 125.0 20.6 90 116.2 25.4 98.1 17.8 105 99.5 23.4 84.2 17.7120 85.6 22.5 69.8 15.6 150 67.2 18.0 57.2 11.4 180 57.7 17.3 54.1 12.2240 60.4 14.2 72.1 21.4 360 76.7 10.6 114.7 30.8 Composition C produceda shift in the glucose profiles compared to the Humalog ® controlprofile.

Serum insulin concentrations and PK parameters are generated andanalyzed generally as described above, and PK results are provided intable 9 below.

TABLE 9 Tmax Cmax AUC_(INF) CL/F Compound (min) (nM) (min*nM)(mL/min/kg) Humalog ® Mean 64.6 0.703 106 12.3 N = 13 SE 9.21 0.09559.10 1.09 Median 60.0 0.643 99.5 12.1 Composition C Mean 46.4 0.691 10713.6 N = 11 SE 8.20 0.111 17.6 1.58 Median 45.0 0.540 92.5 13.0 Mean andmedian Tmax are 28% and 25% earlier in Composition C, respectively, thanwith Humalog.Insulin Lispro Formulated with 25 mM Citrate, 0.285 mM Zinc and 25 mMNaCl

A study on compositions comprising 25 mM citrate, zinc and sodiumchloride is performed in diabetic (Alloxan induced), castrated, maleYucatan miniature swine following generally the procedures describedabove.

Test article (Composition D in the table below) is formulated andshipped to the test site, and Humalog® insulin control was a commercialvial on site at test site:

TABLE 10 Name Formulation Composition Composition D 96 U/mL insulinlispro 25 mM citrate 25 mM NaCl 0.285 mM zinc 2.99 mg/mL m-cresol 1.79mg/mL dibasic sodium phosphate 15.2 mg/mL glycerol pH 7.4 Humalog ® 100U/mL KPB 1.88 mg/mL dibasic sodium phosphate 16 mg/mL glycerol 3.15mg/mL meta-cresol 0.3 mM zinc pH 7.4

This study consists of three doses (0.1, 0.2, and 0.4 Units/Kg) ofComposition D compared to Humalog® at the same doses. Study is designedas a 21 pig full cross-over design to study all pigs on all treatments(n=21). For 0.4 Unit/kg dose, two animals were excluded (one for illnessand one for port non-patency) from the Composition D treatment groupyielding n=19 for that treatment group. One animal was excluded fromHumalog® 0.4 U/Kg treatment group (for port non-patency) yielding n=20for that treatment group. All other doses n=21 for each treatment.

Serum glucose data are presented in Table 11 as mean (mg/dL)+/−SEM.

TABLE 11 Humalog ® Composition D Humalog ® Composition D Humalog ®Composition D Time 0.1 U/KG 0.1 U/KG 0.2 U/Kg 0.2 U/Kg 0.4 U/Kg 0.4 U/Kg(min) AVG SEM AVG SEM AVG SEM AVG SEM AVG SEM AVG SEM −30 306.2 7.7310.3 9.3 301.0 7.2 302.3 7.6 319.8 10.0 316.3 11.4 −20 307.7 9.2 312.68.9 305.6 6.8 311.1 8.3 315.7 9.2 313.4 10.2 0 329.9 8.7 329.0 8.8 326.99.3 329.4 9.3 344.5 10.2 341.1 11.5 5 333.5 10.0 340.8 9.2 327.2 9.7330.0 8.3 349.1 10.0 343.1 11.3 10 335.0 12.5 330.1 8.8 332.3 9.9 311.88.6 349.3 10.5 307.9 11.9 15 337.2 11.6 307.3 9.9 323.2 10.5 285.7 9.2335.4 13.4 260.6 12.5 30 317.6 15.9 284.4 13.2 282.2 13.2 236.0 11.0277.3 15.1 182.2 13.6 45 321.0 19.6 293.3 15.8 263.3 15.7 228.8 14.3233.1 17.6 141.3 14.3 60 318.2 22.0 291.9 16.9 231.8 17.7 219.9 17.6184.5 17.1 114.4 14.1 75 303.5 22.8 294.7 18.1 207.7 19.5 208.3 18.2152.4 19.1 96.9 13.7 90 297.7 23.3 289.8 18.8 188.8 18.0 198.0 19.1121.0 17.0 84.6 13.0 105 290.5 22.9 283.9 19.3 167.0 17.1 193.7 19.196.2 16.5 71.1 12.3 120 283.4 22.3 282.3 20.0 149.3 16.6 164.8 18.1 80.117.2 61.1 10.5 150 273.5 21.7 271.4 19.9 124.6 15.6 152.6 17.6 61.3 17.249.8 10.5 180 274.7 17.9 270.7 20.4 113.1 14.4 142.4 16.3 50.6 15.3 43.510.9 240 285.4 16.2 270.2 17.1 124.1 15.5 147.5 17.1 49.1 14.9 45.4 10.5360 302.0 15.8 287.1 17.2 166.9 20.7 176.6 16.2 74.5 21.8 67.4 12.5To statistically compare the change of serum glucose under differentformulations at different time points, derivative analysis, followinggenerally the procedure described above, was utilized. All three dosesof Composition D (0.1, 0.2, and 0.4 U/kg) result in a significantly(p≤0.01) faster decrease of blood glucose level at time points 5, 10 and15 minutes when compared against Humalog® at the same respective dose.

For pharmacokinetic analysis, eleven pigs were chosen at random using anonline randomizing tool. Insulin levels for serum PK samples for thosepigs are measured using a total insulin RIA. Lower and upper limits ofquantitation for the assay are 20 pM and 5000 pM, respectively. Valuesbelow the lower limit of quantitation are assumed to be 20 pM.Non-compartmental pharmacokinetic analyses are performed using PhoenixWinNonlin v6.3.

TABLE 12 Tmax Cmax AUC_(INF) CL/F Compound (min) (nM) (min*nM)(mL/min/kg) 0.1 U/kg Mean 41.8 0.350 44.3 15.3 Humalog ® SE 5.53 0.05315.20 1.62 Median 45 0.283 43.5 13.8 0.1 U/Kg Mean 33.6 0.330 45.3 15.0Composition D SE 15.3 0.0546 5.70 1.60 Median 15 0.231 38.3 15.7 0.2U/Kg Mean 57.3 0.820 111 12.0 Humalog ® SE 7.79 0.195 12.9 1.09 Median60 0.634 106 11.4 0.2 U/Kg Mean 53.6 1.14 132 10.7 Composition D SE 14.60.258 17.5 1.41 Median 30 0.847 115 10.5 0.4 U/Kg Mean 75 1.57 197 13.4Humalog ® SE 11.1 0.435 21.3 1.17 Median 75 0.952 159 15.0 0.4 U/Kg Mean48.6 3.19 327 8.58 Composition D SE 13.2 0.975 46.6 0.961 Median 45 2.16304 7.89

Median Tmax values are 67%, 50% and 40% earlier in the 0.1 U/Kg, 0.2U/Kg and 0.4 U/Kg Composition D groups as compared to those same doseswith Humalog®. Mean Tmax values were 20% and 35% earlier in the 0.1 U/Kgand 0.4 U/Kg Composition D groups as compared to those same doses withHumalog®. Total exposure and Cmax appear greater for Composition D thanHumalog® at the 0.2 U/kg and 0.4 U/kg doses.

Human Insulin, Insulin Aspart and Insulin Glulisine Formulated with 25mM Citrate, and 5 mM MgCl₂

A study on compositions comprising three different insulins, 25 mMcitrate and magnesium chloride is performed in diabetic (Alloxaninduced), castrated, male Yucatan miniature swine following generallythe procedures described above.

Test articles (Compositions E, F and G in the table below) areformulated by adding sufficient citrate and magnesium chloride tocommercial vials of HUMULIN-R®, NOVOLOG® and APIDRA® to reach aconcentration of 25 mM citrate and 5 mM magnesium chloride:

TABLE 13 Name Formulation Composition Novolog ® 100 U/mL insulin aspart0.58 mg/mL NaCl 16 mg/mL glycerol 1.5 mg/mL phenol 1.72 mg/mL m-cresol19.6 μ/mL Zn pH 7.2-7.6 Composition E 97 U/mL insulin aspart 25 mMcitrate 5 mM MgCl2 0.56 mg/mL NaCl 15.5 mg/mL glycerol 1.46 mg/mL phenol1.67 mg/mL m-cresol 19.0 μ/mL Zn pH 7.2-7.6 Humulin-R ® 100 U/mL humaninsulin 16 mg/mL glycerol 2.5 mg/mL m-cresol 0.015 mg/mL Zn pH 7.0-7.8Composition F 97 U/mL human insulin 25 mM citrate 5 mM MgCl2 15.5 mg/mLglycerol 2.43 mg/mL m-cresol 0.015 mg/mL Zn pH 7.0-7.8 Apidra ® 100 U/mLinsulin glulisine 3.15 mg/mL metacresol 6 mg/mL tromethamine 5 mg/mLNaCl 0.01 mg/mL polysorbate 20 pH 7.3 Composition G 97 U/mL insulinglulisine 25 mM citrate 5 mM MgCl2 3.06 mg/mL metacresol 5.8 mg/mLtromethamine 4.85 mg/mL NaCl 0.01 mg/mL polysorbate 20 pH 7.3Study is designed as a full cross-over design to study all pigs on alltreatments (n=20). One animal is excluded from Composition F treatmentgroup for baseline blood glucose <200 mg/dl, resulting in n=19 for thatgroup. Serum glucose data are presented in Table 14 as mean(mg/dL)+/−SEM.

TABLE 14 Time Novolog ® Composition E Humulin-R ® Composition F Apidra ®Composition G (min) AVG SEM AVG SEM AVG SEM AVG SEM AVG SEM AVG SEM −30340.9 8.2 338.1 5.8 313.2 7.7 319.7 8.5 298.3 8.4 287.9 6.4 −20 353.77.8 348.9 6.7 324.2 7.7 325.7 8.2 310.4 9.1 299.0 6.5 0 363.4 9.5 361.28.2 334.1 10.3 337.5 10.2 319.5 10.6 302.9 8.5 5 377.1 10.4 374.3 8.7346.9 10.4 350.6 10.9 329.6 10.7 309.6 9.5 10 378.4 11.2 362.8 10.1355.5 10.6 342.5 12.2 334.7 11.9 304.2 10.7 15 380.8 12.5 343.9 12.4360.3 10.9 340.8 14.2 331.7 13.2 290.2 12.8 30 362.1 16.1 294.4 18.8367.4 13.8 332.4 17.8 323.8 18.0 257.1 17.3 45 330.6 19.1 269.8 21.6376.9 15.6 328.7 20.0 320.1 20.5 244.4 19.6 60 302.2 22.0 241.6 21.4370.8 17.4 326.9 22.5 304.4 21.3 233.1 19.2 75 285.9 25.2 225.1 21.8360.3 19.1 318.7 22.9 283.1 21.6 223.6 19.3 90 254.1 26.3 205.4 20.4351.0 19.8 306.4 22.9 261.2 21.8 208.3 17.9 105 230.4 26.3 188.0 19.7330.7 19.9 289.6 23.0 239.0 20.9 200.9 18.3 120 196.1 24.8 159.4 17.9308.7 21.1 269.9 22.4 211.1 20.4 181.5 17.6 150 152.9 23.8 140.3 17.6267.3 25.4 216.9 22.5 169.1 20.8 150.5 18.1 180 111.1 20.9 120.0 16.3207.0 22.5 167.7 19.7 130.1 19.1 126.7 16.8 240 71.4 12.7 107.3 17.7130.7 21.1 107.6 15.9 84.5 14.3 79.4 11.6 360 65.8 8.5 90.4 16.9 70.417.9 45.1 8.0 62.6 10.4 50.5 5.8

The citrate-containing formulations of insulin aspart and human insulinproduce a shift in the glucose profiles as compared to Novolog® andHumulin-R® controls, respectively. The citrate-containing formulation ofinsulin glulisine produces a less pronounced shift during approximatelythe first 90 minutes after dosing, as compared to Apidra® control.

Insulin Lispro Formulated with Varying Concentrations of Citrate, MgCl₂,Zinc, Surfactant and NaCl

A study on compositions comprising varying concentrations of citrate,magnesium chloride, zinc, surfactant and/or sodium chloride is performedin diabetic (Alloxan induced), castrated, male Yucatan miniature swinefollowing generally the procedures described above.

Test compositions H and J in the table below are prepared from bulkinsulin lispro active pharmaceutical ingredient (API), and test articlesI and K in the table below are prepared by adding citrate and theindicated stabilizing agents to a vial of Humalog® drug product, andshipped to the test site, and Humalog® insulin control is a commercialvial on site at test site.

TABLE 15 Name Formulation Composition Composition H 97.7 U/mL insulinlispro 25 mM citrate 5 mM MgCl2 0.9 mM zinc 0.09% polysorbate 88 5 mg/mLglycerin Composition I 91.4 U/mL insulin lispro 25 mM citrate 5 mM MgCl215 mM NaCl 0.3 mM Zinc 14.6 mg/mL glycerin Composition J 97.6 U/mLinsulin lispro 15 mM citrate 5 mM MgCl2 15 mM NaCl 0.3 mM zinc 7.6 mg/mLglycerin Composition K 94 U/mL 25 mM citrate 5 mM MgCl2 0.51 mM zinc0.07% w/v P88 15.2 mg/mL glycerin Humalog ® 100 U/mL KPB 1.88 mg/mLdibasic sodium phosphate 16 mg/mL glycerol 3.15 mg/mL meta-cresol 0.3 mMzinc pH 7.4

Study is designed as a full cross-over design to study all pigs on alltreatments (n=19). One animal is excluded from each of the CompositionI, J and K treatment groups for port failure or animal conditionsresulting in n=18 for those treatment groups. Serum glucose data arepresented in Table 16 as mean (mg/dL)+/−SEM.

TABLE 16 Time Humalog ® Composition H Composition I Composition JComposition K (min) AVG SEM AVG SEM AVG SEM AVG SEM AVG SEM −30 372.111.9 374.1 9.6 363.8 10.4 367.8 9.2 384.7 12.4 −20 382.5 11.7 376.9 8.9378.6 11.2 380.2 10.4 398.2 12.6 0 399.9 12.8 402.8 10.6 399.2 11.0398.3 10.0 409.6 11.7 5 409.7 12.1 412.6 10.2 410.8 11.0 408.3 10.3423.8 11.2 10 412.1 13.6 395.8 11.3 387.2 10.5 392.4 10.8 406.0 11.5 15410.2 13.4 369.3 14.1 352.2 12.0 361.8 14.6 376.8 13.5 30 377.2 18.1339.2 16.1 295.2 18.4 317.4 20.8 324.4 16.9 45 353.8 21.7 321.0 18.5280.1 24.4 299.8 24.3 306.3 21.7 60 331.6 25.2 307.1 23.2 271.3 25.5289.2 26.8 289.2 22.3 75 298.4 27.9 288.5 24.5 258.3 25.1 272.0 28.2275.4 22.2 90 279.7 28.9 275.9 25.9 245.2 23.6 258.1 28.4 265.7 22.1 105256.1 28.5 258.6 26.2 235.4 23.6 248.3 27.0 251.1 20.4 120 242.3 28.2252.1 27.9 227.2 22.8 240.7 26.4 242.5 21.0 150 221.7 26.5 228.5 27.5216.9 21.4 227.8 23.9 221.9 20.4 180 207.3 25.4 224.1 27.3 204.6 21.3221.1 20.6 215.8 21.8 240 214.7 24.1 211.5 27.4 191.9 21.7 204.6 18.4209.4 20.3 360 185.2 28.7 160.4 24.9 162.9 19.9 153.2 19.9 151.7 22.3

The citrate-containing formulations produce a shift in the glucoseprofiles as compared to Humalog® controls.

Serum insulin concentrations and PK parameters for Humalog® andCompositions H, J and K are generated and analyzed generally asdescribed above, and PK results are provided in table 17 below.

TABLE 17 Early ½ Late ½ Tmax Tmax Tmax Tmax spread T50 Formulation (min)(min) (min) (min) (min) Humalog ® Mean 57.2 20.1 161 141 105 SE 5.2 2.819 20 8 Composition H Mean 55.0 9.76 129 119 96.8 SE 11.0 2.07 13 13 6.7Composition J Mean 28.8 9.40 141 131 97.7 SE 5.4 3.58 18 19 6.1Composition K Mean 56.2 16.7 115 98.3 89.3 SE 13.7 6.0 18 19 5.8 Resultsindicate Compositions H, J and K have faster PK onset than Humalog ®.

Stability Studies

Insulin Lispro Formulated with Varying Concentrations of Citrate, SodiumChloride and Magnesium Chloride.

An accelerated shelf-life stability study is performed to assess thestability of insulin lispro when co-formulated with variousconcentrations of citrate, sodium chloride, and, optionally, magnesiumchloride. Stability samples having the compositions set forth in thetable below are prepared either by formulating insulin lispro activepharmaceutical ingredient with the other excipients indicated in thetable below (Compositions L, M and N) or by adding citrate and sodiumchloride to a vial of Humalog® drug product (Composition O).

TABLE 18 Name Formulation Composition Composition L 100 U/mL insulinlispro 35 mM sodium citrate 5 mM MgCl₂ 23 mM NaCl 0.3 mM zinc oxide 3.15mg/mL m-cresol pH 7.5 Composition M 100 U/mL insulin lispro 25 mMcitrate 5 mM MgCl₂ 15 mM NaCl 0.3 mM zinc 3.15 mg/mL m-cresol 4.49 mg/mLglycerol pH 7.5 Composition N 100 U/mL insulin lispro 15 mM citrate 5 mMMgCl₂ 15 mM NaCl 0.3 mM zinc 3.15 mg/mL m-cresol 7.62 mg/mL glycerol pH7.5 Composition O 95 U/mL insulin lispro 25 mM citrate 25 mM NaCl 0.3 mMzinc 1.88 mg/mL dibasic sodium phosphate 3.15 mg/mL m-cresol 16 mg/mLglycerol pH 7.5 Humalog ® 100 U/mL KPB 1.88 mg/mL dibasic sodiumphosphate 16 mg/mL glycerol 3.15 mg/mL meta-cresol 0.3 mM zinc pH 7.4

The solutions are filtered using 0.22 micron PVDF membrane (Cat#SLGV013SL, Millex, Millipore) and distributed into glass vials (Cat#NC4015-1, National Scientific, Thermo Scientific Inc.) with screw-topcaps (Cat# C4015-67A, National Scientific, Thermo Scientific Inc.),incubated at 2 to 8° C. and 30° C. respectively. Samples from the 30° C.incubation temperature were pulled for analysis at initial, 8, 16, 27,36, 44, 54 and 66 day time points. Samples from the 5° C. incubationtemperature are pulled for analysis at initial, 27, 36, 44, 54 and 66day time points.

Size exclusion high-performance liquid chromatography (SEC-HPLC)analysis is performed to assess protein potency and quantify highmolecular weight species in each composition at the stability timepoints using a UV detector at 276 nm. Each sample (10 μL) is separatedat room temperature by using a Sepax Zenix-C SEC-80, 7.8×300 mm, 3 μmparticles column (catalog# 233080-7830) at a flow rate of 1.0 mL /minutewith isocratic elution of mobile phase (0.1% TFA, 50% ACN) over a runtime of 25 minutes. Insulin concentrations are calculated by comparingthe insulin peak area to an insulin lispro standard area and adjustingfor purity determined by reverse phase HPLC. Results, reported in IU/mL,are given below.

TABLE 19 30° C. incubation temperature Day Day Day Day Day Day Day Day 08 16 27 36 44 54 66 Composition L 102.26 100.86 101.14 102.33 99.43100.71 101.23 98.57 Composition M 102.83 102.46 100.06 104.37 100.48100.99 101.29 99.99 Composition N 102.95 102.34 101.09 104.75 101.52101.93 100.18 99.98 Composition O 93.44 94.24 92.49 97.27 91.20 91.6590.83 89.96 Humalog ® 99.82 100.43 97.66 103.13 97.71 96.73 98.38 97.19

TABLE 20 4° C. incubation temperature Day Day Day Day Day Day 0 27 36 4454 66 Composition L 102.26 104.63 102.93 102.87 105.14 102.59Composition M 102.83 105.93 103.45 103.63 103.04 99.56 Composition N102.95 106.56 103.25 102.83 102.69 104.28 Composition O 93.44 94.7892.50 91.40 93.88 95.87 Humalog ® 99.82 103.94 97.82 99.76 97.92 99.74Insulin loss for citrate-containing and Humalog ® control samples isless than 5% for all samples out to 66 days at 4° C. and 30° C.

Percentage of high molecule weight (% HMW) is calculated by integratingthe total area % of all peaks eluting prior to the main peak. Results (%HMW) are given in the tables below.

TABLE 21 30° C. incubation temperature Day Day Day Day Day Day Day Day 08 16 27 36 44 54 66 Composition L 0.18 0.20 0.27 0.37 0.40 0.45 0.500.63 Composition M 0.19 0.21 0.25 0.32 0.35 0.39 0.45 0.58 Composition N0.16 0.21 0.25 0.32 0.36 0.43 0.46 0.57 Composition O 0.29 0.35 0.440.53 0.62 0.65 0.77 0.87 Humalog ® 0.30 0.34 0.43 0.57 0.65 0.69 0.810.97

TABLE 22 4° C. incubation temperature Day Day Day Day Day Day 0 27 36 4454 66 Composition L 0.18 0.19 0.20 0.15 0.18 0.20 Composition M 0.190.17 0.18 0.15 0.19 0.19 Composition N 0.16 0.18 0.16 0.19 0.16 0.17Composition O 0.29 0.34 0.36 0.34 0.31 0.35 Humalog ® 0.30 0.34 0.350.34 0.32 0.35 HMW formation is less than 1% for all citrate-containingand Humalog ® control samples out to 66 days at 4° C. and 30° C.

Reversed phase high-performance liquid chromatography (RP-HPLC) analysisis performed to assess protein purity in each composition at thestability time points using a UV detector at 214 nm. Each sample (5 μL)is separated at 40° C. by using a Waters BioSuite C18 PA-B, 3.5 μm,2.1×150 mm HPLC column (Part #186002435), or comparable column, at aflow rate of 0.6 mL/minute with mobile phase A (50 mM sulfate, pH2.3+20% acetonitrile (v/v)) and mobile phase B (50 mM sulfate, pH2.3+50% acetonitrile (v/v)). Gradient of mobile phase B at 0, 3, 15, 21,26, 27, 27.5 and 35.0 min is 21, 25, 25, 30, 80, 80, 21 and 21%,respectively. Percentage of sample outside of main peak and A-21desamido is determined by subtracting the main peak percentage and theA-21 desamido percentage from 100 percent. Collectively, these peaks areconsidered other related substances (ORS). The results (% outside ofmain peak and A-21 and desamido) are given below.

TABLE 23 30° C. incubation temperature Day Day Day Day Day Day Day Day 08 16 27 36 44 54 66 Comp. L 0.52 0.71 0.96 1.20 1.69 1.65 2.03 2.25Comp. M 0.57 0.83 1.00 1.20 1.70 1.63 2.02 2.18 Comp. N 0.50 0.87 1.121.28 1.82 1.72 2.01 2.25 Comp. O 0.78 1.18 1.49 1.63 1.89 2.14 2.53 2.78Humalog ® 0.74 1.15 1.47 1.68 1.77 2.20 2.63 2.97

TABLE 24 4° C. incubation temperature Day Day Day Day Day 0 27 44 54 66Composition L 0.52 0.62 0.59 0.66 0.68 Composition M 0.57 0.67 0.60 0.630.73 Composition N 0.50 0.66 0.59 0.67 0.76 Composition O 0.78 1.08 0.890.99 0.94 Humalog ® 0.74 1.05 0.87 0.95 0.91 The ORS for allcitrate-containing and Humalog ® control samples is less than 1.5% at 4°C. for 66 days and less than 3.0% at 30° C. for 66 days.Physical Stability of Insulin Lispro Formulated with 25 mM Citrate, 0.3mM Zinc, 5 mM MgCl₂, 0.09% Poloxamer and Varying Chloride Concentrations

A lispro concentrate stock formulation is prepared comprising 200 U/mLinsulin lispro/mL, 32 mg glycerin/mL, 6.30 mg metacresol/mL, 0.6 mM zincin water for bulk sterile operations. The formulation is pH adjustedwith hydrochloric acid to dissolve the insulin lispro and then adjustedwith sodium hydroxide to pH of 7.45.

Test formulations are then prepared by diluting the insulin lisproconcentrate with sodium citrate buffer, magnesium chloride hexahydratepowder, poloxamer 188 solution, and granular sodium chloride and q.s.with water for bulk sterile operations to a composition of 25 mMcitrate, 5 mM MgCl₂, 0.09% poloxamer 188, 100 units/mL lispro, 16 mg/mLglycerin, 3.15 mg/mL Metacresol, and 0.3 mM zinc with either 23.2(Composition P) or 17.7 (Composition Q) mM total chloride.

Formulations are sterile filtered and then volumetrically transferred to10 mL glass vials with a 7 mL fill, stoppered, and crimp sealed.

In a thirteen day accelerated stability study (30° C., shaken 75 atstrokes per minute (spm)) vials undergo visual inspection on days 2, 4,8 and 13. Vials containing a clear and colorless solution with noprecipitate are considered scored as a “pass,” while a vial containingparticulates or a solution which is not clear and/or discolored isrecorded as “fail.” Data are provided below in Table 25.

TABLE 25 Day 2 Day 4 Day 8 Day 13 Composition (n = 2) (n = 2) (n = 4) (n= 4) Composition P 100% Pass 100% Pass 100% Pass 50% pass Composition Q100% Pass 100% Pass 100% Pass  0% passPhysical Stability of Insulin Lispro Formulated with 25 mM Citrate, 0.3mM Zinc, 0.09% Poloxamer and 23 mM Total Chloride With and WithoutMagnesium

A lispro concentrate stock formulation is prepared comprising 200 U/mLinsulin lispro, 32 mg glycerin/mL, 6.30 mg metacresol/mL, and 0.6 mMzinc in water for bulk sterile operations. The formulation is pHadjusted with hydrochloric acid to dissolve the insulin lispro and thenadjusted with sodium hydroxide to pH of 7.45.

Test formulations are prepared by diluting the lispro concentrate withsodium citrate buffer, poloxamer 188 solution, granular sodium chloride,optionally, with magnesium chloride hexahydrate powder, and q.s. withwater for bulk sterile operations to compositions of 25 mM citrate,0.09% w/v poloxamer 188, 100 units/mL lispro, 16 mg/mL glycerin, 3.15mg/mL Metacresol, 0.3 mM zinc, 23 mM total chloride and either 0(Composition R) or 5 (Composition S) mM MgCl₂.

Test formulations are sterile filtered and then volumetricallytransferred to 10 mL glass vials with a 7 mL fill, stoppered, and crimpsealed.

Vials are subjected to a thirteen day accelerated stability study withperiodic visual inspection following generally the method describedabove. Data are provided below in Table 26.

TABLE 26 Day 2 Day 4 Day 8 Day 10 Day 13 Composition (n = 2) (n = 2) (n= 4) (n = 4) (n = 4) S 100% Pass 100% Pass 100% Pass 100% pass 50% passR 100% Pass  50% Pass  0% Pass N/A N/A Results support that magnesiumimproved the stability of Composition S relative to Composition R.Physical Stability of Insulin Lispro Formulated with 25 mM Citrate, 5 mMMgCl₂, 20 mM Chloride, 0.6 mM Zinc, 0.09% Poloxamer

A 69 day accelerated shelf life study is conducted on a formulationcomprising 100 U/mL insulin lispro, 5 mM MgCl₂, 20 mM chloride, 0.6 mMzinc, 0.09% w/v poloxamer 188, 5 mg/mL glycerin and 3.15 mg/mL m-cresol(Composition T).

A bulk concentrate formulation is prepared by dissolving of glycerin,metacresol, lispro and zinc oxide in water for bulk sterile operationsby addition of HCl to lower the pH. After adjustment of pH to 7.4-7.5with NaOH, a solution of appropriate concentration of citrate andadditional stabilizing agents to achieve the final target excipientconcentration of Composition T is added. This solution is made up ofsodium citrate buffer, zinc chloride, magnesium chloride hexahydrate,and poloxamer 188 in water for bulk sterile operations. After adjustmentof pH to 7.4-7.5, if needed, sodium chloride solution is added toachieve target total chloride concentration. The formulation is thenq.s. to volume with water for bulk sterile operations.

Formulations are sterile filtered and then volumetrically transferred to10 mL glass vials with a 10.3 mL fill, stoppered, and crimp sealed.Vials are stored static and upright at 30° C. and visual inspected,following generally the method described above, at days 36 and 69. Allvials inspected at each timepoint pass (n=3 for day 36 and n=5 for day69). These results support that Composition T remains clear andcolorless with no precipitate in an accelerated shelf life study.

Insulin Lispro Formulated with Varying Concentrations of Citrate,Magnesium, Chloride, Zinc, Surfactant, Glycerin and TRIS

Physical stability of the following seven formulations is tested in a32-day simulated patient in use study:

TABLE 27 Citrate MgCl₂, Total Cl— Zn, P88 Glycerin TRIS, m-cresolComposition (mM) (mM) (mM) (mM) (% w/v) (mg/mL) (mM) (mg/mL) U 25 5 210.6 0.12 5 0 3.15 V 25 5 21 0.9 0.09 5 0 3.15 W 25 8.3 31 0.6 0.06 5 03.15 X 20 5 20 0.6 0.09 5 0 3.15 Y 20 5 20 0.6 0.12 5 0 3.15 Z 25 5 >300.6 0.09 1.3 40 3.15 AA 25 8.3 28 0.6 0.09 4.1 10 3.15

Concentrate lispro stock solutions are prepared by dissolvingappropriate quantities of glycerin, metacresol, lispro, zinc oxide and,optionally, TRIS in water for bulk sterile operations by addition of HClto lower the pH. Compositions of the two concentrate solutions are asfollows: (1) 200 U/mL lispro, 12 mM total chloride, 10 mg/mL glycerin,6.3 mg/mL Metacresol, 0.6 mM total zinc; and (2) 200 U/mL lispro, 51 mMtotal chloride, 2.6 mg/mL glycerin, 6.3 mg/mL Metacresol, 0.6 mM totalzinc, 80 mM TRIS. The solutions are then pH adjusted to 7.4-7.5 withNaOH or HCl and filled to volume with water for bulk sterile operations.

Test compositions are prepared by diluting the lispro stock solutions bysequential addition of sodium citrate buffer, zinc in HCl solution,sodium hydroxide to adjust pH, magnesium chloride hexahydrate solution,poloxamer 188 solution, and sodium chloride solution. Compositions aresterile filtered and then volumetrically q.s. to volume with water forbulk sterile operations. Compositions are volumetrically transferred to10 mL glass vials with a 10.3 mL fill, stoppered, and crimp sealed.

Compositions are stored at 30° C. and subjected to simulated in-usedosing conditions 3 times a day, as described in the following steps:obtain an insulin syringe and withdraw 8 units of air; insert the needleinto the vial while the vial is in the upright position; ensure theneedle tip does not touch the insulin solution; inject the air into thevial; while keeping the needle in the vial, turn the vial and syringeupside down; withdraw 8 units of the composition; if there are airbubbles in the syringe slowly move the plunger to push the bubble(s)back into the vial; adjust the syringe plunger so the final dosage is 8units; remove the syringe and place vials back into 30° C. incubator.

Visual inspections, following generally the method described above, areconducted on days 15, 20, 25, 28 and 32. Data are provided in Table 28.

TABLE 28 Com- posi- 15 Days 20 Days 25 Days 28 Days 32 Days tion (n = 5)(n = 5) (n = 5) (n = 5) (n = 5) U 100% Pass 100% Pass 100% Pass 80% Pass80% Pass V 100% Pass 100% Pass 100% Pass 100% Pass 100% Pass W 100% Pass100% Pass 100% Pass 100% Pass 100% Pass X 100% Pass 100% Pass 100% Pass100% Pass 100% Pass Y 100% Pass 100% Pass 100% Pass 80% Pass 67% Pass Z100% Pass 100% Pass 60% Pass 10% Pass 0% Pass AA 100% Pass  80% Pass 60%Pass 20% Pass 0% Pass

Collectively, the studies described above demonstrate that compositionsof insulin with certain specific concentrations of citrate and otherexcipients such as zinc, magnesium, chloride and surfactant have earlieronset of action, as well as earlier glucose lowering effects, and incertain embodiments are chemically and physically stable.

Sequences Human insulin A-chainGly Ile Val Glu Gln Cys Cys Thr Ser Ile Cys SerLeu Tyr Gln Leu Glu Asn Tyr Cys Asn (SEQ ID NO: 1).Human insulin B-chain Phe Val Asn Gln His Leu Cys Gly Ser His Leu ValGlu Ala Leu Tyr Leu Val Cys Gly Glu Arg Gly PhePhe Tyr Thr Pro Lys Thr (SEQ ID NO: 2). Insulin lispro B-chainPhe Val Asn Gln His Leu Cys Gly Ser His Leu ValGlu Ala Leu Tyr Leu Val Cys Gly Glu Arg Gly PhePhe Tyr Thr Lys Pro Thr (SEQ ID NO: 3). Insulin aspart B-chainPhe Val Asn Gln His Leu Cys Gly Ser His Leu ValGlu Ala Leu Tyr Leu Val Cys Gly Glu Arg Gly PhePhe Tyr Thr Asp Lys Thr (SEQ ID NO: 4). Insulin glulisine B-chainPhe Val Lys Gln His Leu Cys Gly Ser His Leu ValGlu Ala Leu Tyr Leu Val Cys Gly Glu Arg Gly PhePhe Tyr Thr Pro Glu Thr (SEQ ID NO: 5).

We claim:
 1. A pharmaceutical composition comprising: a. an insulin; b.citrate, in a concentration from about 10 to about 30 mM; c. zinc, in aconcentration from about 0.2 to about 2 mM; d. magnesium, in aconcentration from about 1 to about 15 mM; e. total chloride, in aconcentration from about 10 to about 60 mM; f. a surfactant, in aconcentration from about 0.001 to about 0.2% w/v; and g. a preservative.2. The pharmaceutical composition of claim 1, wherein the insulin isinsulin lispro.
 3. The pharmaceutical composition of claim 2 wherein theinsulin lispro concentration is from about 100 to about 500 U/mL.
 4. Thepharmaceutical composition of claim 3 wherein the concentration ofcitrate is from about 15 to about 25 mM.
 5. The pharmaceuticalcomposition of any of claim 4 wherein the concentration of zinc is fromabout 0.3 to about 1.1 mM.
 6. The pharmaceutical composition of claim 1wherein the magnesium concentration is provided through the inclusion ofmagnesium chloride.
 7. The pharmaceutical composition of claim 6 whereinthe magnesium concentration is from about 3 to about 8 mM.
 8. Thepharmaceutical composition of claim 1 wherein the chloride concentrationis from about 15 to about 35 mM.
 9. The pharmaceutical composition ofclaim 8 wherein the chloride concentration is from about 20 to about 25mM.
 10. The pharmaceutical composition of claim 1 comprising sodiumchloride in a concentration ranging from about 1 to about 50 mM.
 11. Thepharmaceutical composition of claim 10 comprising sodium chloride in aconcentration ranging from about 1 to about 25 mM.
 12. Thepharmaceutical composition of any of claim 11 comprising sodium chloridein a concentration ranging from about 1 to about 20 mM.
 13. Thepharmaceutical composition of claim 1 wherein the surfactant ispoloxamer
 188. 14. The pharmaceutical composition of claim 13 whereinthe concentration of poloxamer 188 is from about 0.003 to about 0.2%w/v.
 15. The pharmaceutical composition of claim 14 wherein theconcentration of poloxamer 188 is from about 0.03 to about 0.12% w/v.16. The pharmaceutical composition of claim 15 wherein the concentrationof poloxamer 188 is from about 0.06 to about 0.09% w/v.
 17. Thepharmaceutical composition of claim 1, wherein the preservative isselected from the group consisting of phenol and metacresol and mixturesthereof.
 18. The pharmaceutical composition of claim 17, wherein thepreservative is metacresol.
 19. The pharmaceutical composition of claim18, wherein the metacresol concentration is from about 2.5 to about 3.8mg/mL.
 20. A pharmaceutical composition comprising: a. insulin lispro,in a concentration from about 100 to about 200 IU/mL; b. citrate, in aconcentration from about 15 to about 25 mM; c. magnesium chloride, in aconcentration of about 3 to about 8 mM; d. zinc, in a concentration fromabout 0.6 to about 0.9 mM; e. total chloride, in a concentration fromabout 20 to about 25 mM; f. poloxamer 188, in a concentration from about0.06 to about 0.09% w/v; and g. metacresol, in a concentration of about2.8 to about 3.5 mg/mL; and wherein the pH of the composition is fromabout 7.0 to 7.8.
 21. A pharmaceutical composition comprising: a.insulin lispro, in a concentration of 100 IU/mL; b. citrate, in aconcentration of about 25 mM; c. zinc, in a concentration of about 0.9mM; d. magnesium chloride, in a concentration of about 5 mM; e. totalchloride, in a concentration of about 20 to about 25 mM; f. poloxamer188, in a concentration of about 0.09% w/v; and g. metacresol, in aconcentration of about 3.15 mg/mL; and wherein the pH of the compositionis about 7.4 mM.
 22. A pharmaceutical composition comprising: a. insulinlispro, in a concentration of 200 IU/mL; b. citrate, in a concentrationfrom about 15 to about 25 mM; c. magnesium chloride, in a concentrationof about 3 to about 8 mM; d. zinc, in a concentration from about 0.6 toabout 0.9 mM; e. total chloride, in a concentration from about 20 toabout 25 mM; f. poloxamer 188, in a concentration from about 0.06 toabout 0.09% w/v; and g. metacresol, in a concentration of about 2.8 toabout 3.5 mg/mL; and h. wherein the pH of the composition is from about7.0 to 7.8.
 23. A pharmaceutical composition comprising: a. an insulin,in a concentration of about 100 to about 200 IU/mL; b. citrate, in aconcentration from about 15 to about 35 mM; c. magnesium chloride, in aconcentration of up to about 5 mM; d. zinc, in a concentration fromabout 0.2 to about 0.8 mM; e. sodium chloride, in a concentration fromabout 15 to about 25 mM; f. a surfactant; g. metacresol, in aconcentration of about 2.8 to about 3.5 mg/mL; and h. wherein the pH ofthe composition is from about 7.0 to 7.8.
 24. The pharmaceuticalcomposition of claim 1, wherein the composition provides for an uptakeof insulin into the blood that is at least 20% more rapid than forcompositions which contain the same insulin but which do not containcitrate.
 25. The pharmaceutical composition of any of claim 1, whereinthe composition is stable to allow for storage of at least 24 months at2-8° C. and up to 28 days in-use at temperatures of up to 30° C.
 26. Thepharmaceutical composition of claim 1, wherein the composition does notinclude EDTA.
 27. The pharmaceutical composition of claim 1, wherein thecomposition does not include any vasodilatory agent.
 28. Thepharmaceutical composition of claim 1, wherein the composition does notinclude any oligosaccharides.
 29. A method of treating diabetescomprising administering to a human in need thereof an effective dose ofthe pharmaceutical composition of claim 1.